CN1668744A - Method of transforming soybean - Google Patents

Method of transforming soybean Download PDF

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Publication number
CN1668744A
CN1668744A CN 03816844 CN03816844A CN1668744A CN 1668744 A CN1668744 A CN 1668744A CN 03816844 CN03816844 CN 03816844 CN 03816844 A CN03816844 A CN 03816844A CN 1668744 A CN1668744 A CN 1668744A
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soybean
method
explants
primary
agrobacterium
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CN 03816844
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R·柯翰
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辛根塔参与股份公司
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205Agrobacterium mediated transformation

Abstract

本公开提供了农杆菌(Agrobacterium)介导的转化大豆细胞或组织以及将转化细胞和组织再生为转化植物的方法。 The present disclosure provides an Agrobacterium (of Agrobacterium) transformation of soybean mediated cell or tissue and the transformed plant into a cell and tissue regeneration. 这些方法可用于转化许多种大豆栽培品种。 These methods can be used to transform a number of soybean cultivars.

Description

转化大豆的方法 Soybean transformation methods

相关申请的交叉参考本申请要求2002年6月22日提交的序列号NO.60/390,562的美国临时申请的利益,该申请的全部内容以参考文献的方式并入本文。 CROSS REFERENCE TO RELATED APPLICATIONS This application claims the benefit of US Provisional Application Serial Number NO.60 / 390,562 of June 22, 2002 submitted by the interests of the entire contents of which application is incorporated herein by reference.

发明领域本发明一般涉及植物转化的方法,更具体地,涉及转化大豆细胞和组织的方法。 Field of the Invention The present invention relates generally to methods of plant transformation, and more particularly, to a method of transforming soybean cells and tissues. 本发明还涉及从转化的大豆细胞和组织再生转基因大豆植物的方法。 The present invention further relates to a method for regenerating a transgenic soybean plants from the transformed soybean cells and tissues. 本发明还涉及这些方法获得的转基因大豆植物和种子。 The present invention further relates to transgenic soybean plants and seeds obtained by these methods.

发明背景大豆是一种主要的食物和饲料来源,其在全球种植的土地比任何其它的双子叶作物都多。 Background of the Invention Soy is a major source of food and feed, its planted land in the world than any other dicotyledonous crops. 据报导大豆种植超过5000万公顷面积。 It was reported that more than 50 million hectares of soybean planted area. 遗憾地是,在美国仅仅少数的植物引进产生了大豆主要的栽培变种,因而,这一窄的种质基础限制了大豆育种的潜能。 Unfortunately, in the United States to introduce a small number of plants produce only major soybean cultivars, therefore, this narrow germplasm base limits the potential for soybean breeding. 栽培的大豆品种中这一有限的遗传基础限制了用传统育种方法开发具有改良或价值增值性状的变种的能力。 Soybean varieties cultivated in this limited genetic base have limited the ability to develop improved varieties or value-added traits using traditional breeding methods.

因此采用遗传工程技术修饰大豆可以以传统育种方法或组织培养诱导变异难以达到的方式促进新品种的开发,例如具有抗除草剂、抗病(例如抗病毒)和种子质量改良特性的新品种。 Thus modified using genetic engineering techniques can be cultured in a conventional soybean breeding methods or difficult to reach tissue inducing mutation approach facilitated the development of new varieties, such as new varieties having herbicide resistance, disease resistance (e.g. antiviral) seed quality and improved properties.

开发有效的转化系统对于植物中基因表达的分析是必须的。 Develop effective transformation system for the analysis of gene expression in plants is a must. 这种系统的要求包括合适的靶植物组织以允许有效的植物再生,基因传输工具以有效地传送外源DNA进入靶植物细胞,以及有效的方法以选择转化的细胞。 Requirement of such systems include a suitable target plant tissue to allow efficient plant regeneration, gene transfer tool to effectively transfer DNA into target plant an exogenous cells, and effective way to select transformed cells. 在双子叶种类的遗传转化中,经常使用利用根癌农杆菌(Agrobacterium tumefaciens)的转化系统作为基因传输工具。 In the genetic transformation of dicotyledonous species, often using Agrobacterium tumefaciens transformation system (Agrobacterium tumefaciens) as a tool for gene delivery. 目前,用于农杆菌介导转化的优选靶组织包括子叶、叶组织和下胚轴。 Currently, preferred target tissues for transformation include Agrobacterium-mediated cotyledon, hypocotyl and leaf tissue. 高速微粒轰击为基因传输入双子叶植物提供了可供选择的方法。 High-speed particle bombardment gene transfer input dicots provides alternative methods.

大豆中农杆菌介导的基因传输远非是常规的。 Soybean gene transfer mediated by Agrobacterium far is conventional. 在公众可获得的报告中,经常提到分生组织和子叶组织作为用于农杆菌介导基因传输的靶子。 In the publicly available reports, often referred to meristematic and cotyledon tissue as targets for Agrobacterium-mediated gene transfer. 不过,经常不能从这两个外植体资源达到可靠和有效的转化和再生。 However, often these two explants can achieve a reliable and resource efficient transformation and regeneration.

Chee等的美国专利号NO.5,169,770和5,376,543讨论了转化大豆产生转基因植物的非组织培养方法,其中培养种子发芽,然后用细菌细胞特别是农杆菌菌株接种分生组织或中胚层的细胞组织,细菌细胞通过感染将DNA转移到外植体中。 Chee et al and U.S. Patent No. 5,376,543 discussed NO.5,169,770 non-transformed soybean tissue culture methods of producing a transgenic plant, wherein the culture seed germination, and then the bacterial cells, particularly tissue cells inoculated Agrobacterium strain meristem or mesodermal, bacteria cells infected by DNA transfer into the explants. 该方法依赖于预先形成的新芽的生长。 The method relies on the growth of pre-formed shoots.

Parrot WA等(1989),“大豆初级转化体的回收,”PlantCell Reports 7:615-617,报导了与农杆菌共培养后从未成熟的子叶组织中回收大豆转化体。 (1989), "recovered primary transformants soybean," Parrot WA et PlantCell Reports 7: 615-617, reported the Agrobacterium co-cultivation of cotyledon tissue from immature soybean transformants were recovered. 不过,这些再生的植物是嵌合的,转移基因不能遗传给子代。 However, these regenerated plants are chimeric, gene transfer can not be passed on to offspring.

美国专利号NO.5,416,011(Hinchee等)讨论了利用要求去除下胚轴、保留并分开子叶的子叶外植体,并用含有目的基因的根癌农杆菌载体接种该子叶外植体以插入嵌合基因。 U.S. Patent No. NO.5,416,011 (Hinchee etc.) discusses requirements removed using hypocotyls and retain the cotyledons separate cotyledon explants and cotyledon explants inoculated with Agrobacterium tumefaciens vector containing the gene to insert chimeric genes .

Yan B.等(2000),“利用未成熟合子子叶外植体根癌农杆菌介导的大豆转化,”Plant Cell Reports 19:1090-1097,报导用未成熟子叶农杆菌介导的大豆转化中总的转化频率为0.03%。 Yan B. et al. (2000), "soybean transformation using immature zygotic cotyledonary explants with Agrobacterium tumefaciens mediated," Plant Cell Reports 19: 1090-1097, reported that use of immature cotyledons soybean transformation Agrobacterium mediated the total conversion frequency of 0.03%.

Martinell等的美国专利号NO.6,384,301描述了农杆菌介导基因传输入分离的大豆胚轴分生组织细胞中。 U.S. Patent No. Martinell et NO.6,384,301 described Agrobacterium-mediated gene transfer input isolated soybean hypocotyl meristem cells. 这些方法不包括愈伤组织期组织培养。 These methods do not include callus tissue culture.

从以上描述的工作来看,显然地,当利用分生组织和子叶组织作为用于农杆菌介导的基因传输的资源外植体时,相关的工作者很少实现建立一个可靠的大豆转化系统的目标。 From the work described above, it is clear, when the use of meristematic and cotyledon tissue is used as an external resource for Agrobacterium-mediated gene transfer of the explants, little relevant workers achieved for a reliable soybean transformation system The goal. 因此,为了开发更有效的大豆转化系统,需要继续探索新的方法,包括新的资源外植体。 Therefore, in order to develop a more efficient soybean transformation system, we need to continue to explore new methods, including new resources explants.

已经证明,在大豆组织培养中可以从上胚轴组织和初生叶组织获得植物再生。 Has proven, plant regeneration can be obtained from hypocotyl tissue and primary leaf tissue in tissue culture soybeans. 不过,至今为止,在大豆中用这两种外植体源作为基因传输的靶子还没有成功转化的报导。 However, so far, with both soybean explants source reported as a target gene transfer has not been successfully transformed.

Wright MS等(1987)“Glycine max L.Merr.的起始和繁殖:来自组织培养的上胚轴的植物,”Plant Cell Reports 8:83-90,描述了芽从大豆上胚轴组织成功的起始和增殖。 Wright MS, etc. (1987) ". Glycine max L.Merr initiation and propagation: from the epicotyl tissue culture plants," Plant Cell Reports 8: 83-90, describes the shoots from the soybean hypocotyl tissue success initiation and proliferation. 在含有20μM细胞分裂素的Schenk和Hildebrandt培养基中培养5周诱导外植体上胚轴形成芽。 Schenk and Hildebrandt cultured in medium containing 20μM cytokinin bud formation on the hypocotyl explant induction of five weeks. 在含有2.1nM picloram和0.1μM苄基腺嘌呤的培养基中芽保持增殖。 In media containing 2.1nM picloram and 0.1μM benzyladenine holding the bud proliferation.

Wright MS等(1987)“大豆(Glycine max L.Merr.)从培养的初生叶组织中再生,”Plant Cell Reports 6:83-89,描述了从27种大豆的初生叶组织中再生植物的可重复的方法。 Wright MS et al. (1987) "Soybean (. Glycine max L.Merr) primary leaves from regenerated tissue culture," Plant Cell Reports 6: 83-89, describes a primary regeneration of plants from the 27 kinds of leaf tissue of soybean repeatable method. 他们发现尽管已证明2,4,5-三氯苯氧基乙酸对于再生是必须的,但发现苄基腺嘌呤(BA)能增强再生。 They found that despite proven 2,4,5-trichlorophenoxyacetic acid for regeneration is necessary, but found benzyladenine (BA) to enhance regeneration.

Rajasekaren K.等(1997)“培养的大豆(Glycine max L.Merr)上胚轴和初生叶的体细胞胚胎发生,”In Vitro Cellular &Developmental Biology 33(2):88-91,描述了几种大豆通过体细胞胚胎发生从温室栽培植物的未成熟种子的培养上胚轴和初生叶组织的再生。 Rajasekaren K. et al. (1997) "cultured soybean hypocotyls (Glycine max L.Merr) primary leaf and somatic embryogenesis occurs," In Vitro Cellular & amp; Developmental Biology 33 (2): 88-91, describes several regeneration from hypocotyl cultured immature seeds of cultivated plants in the greenhouse and soybean primary leaf tissue via somatic embryogenesis. 他们发现当具有完整合子胚胎轴的半子叶在补充了46.2μM2,4-D的Murashige和Skoog(MS)培养基上培养时,可从上胚轴和初生叶诱导体细胞胚胎发生。 They found that when the half zygotic embryos with intact cotyledons shaft supplemented when cultured on Murashige 46.2μM2,4-D and Skoog (MS) medium, can induce somatic embryogenesis from the epicotyl and the primary leaf. 在没有与半子叶培养的情况下,从提取的胚轴、上胚轴或初生叶中没有观察到胚胎发生。 Cultured in the absence of the half-cotyledons, hypocotyls from the extract, or epicotyl leaves was not observed primary embryogenesis. 在含有11.3μM苄基腺嘌呤的Cheng氏基础培养基上,可从发芽体细胞胚胎获得芽尖的快速增殖。 Based on Cheng's medium containing 11.3μM benzyladenine, can be obtained rapidly proliferating shoot tips from germinating somatic embryos.

发明概述本发明提供了转化大豆细胞和将转化细胞再生为转化植物的方法。 SUMMARY The present invention provides a method for transforming a transformed plant cell and the transformed soybean cell regeneration. 所述方法可用于转化多种大豆栽培品种。 The method can be used to transform a variety of soybean cultivar.

本发明提供了一种新的大豆外植体,其使根癌农杆菌能高效地介导基因传输至大豆细胞中。 The present invention provides a new soybean explants, which makes possible to efficiently Agrobacterium tumefaciens-mediated gene transfer into soybean cells.

具体地,本发明提供了转化大豆细胞或组织的方法,所述方法包括:(a)通过以下步骤从大豆种子制备外植体: In particular, the present invention provides a method of transforming soybean cells or tissue, said method comprising: (a) from soybean seeds explants prepared by the following steps:

(i)从所说的种子中去除全部或部分下胚轴;(ii)从所说的种子中去除一个子叶及其邻近的腋芽(axillary bud),并且保留一个其上附有上胚轴和初生叶的子叶;(iii)去除保留子叶的部分初生叶,由此产生初生叶基部;以及(b)将该外植体和含有待掺入到大豆细胞基因组中的目的核酸的农杆菌共培养。 (I) is removed from said seeds all or part of hypocotyls; (ii) removing a cotyledon and the adjacent axillary buds (axillary bud) from said seed, and retained thereon with a epicotyl and cotyledons primary leaves; (iii) removing retained portions of the cotyledons primary leaf, thereby producing primary leaf base; and (b) the explant and Agrobacterium containing the nucleic acid to be incorporated into the genome of soybean cells co-cultured .

在另外的实施方案中,所述方法还包括一个或多个下述内容:从初生叶基部及其邻近的上胚轴诱导芽形成;在含有选择剂的培养基中培养该芽;选择转化的芽;以及从转化的芽再生转化的植物。 In further embodiments, the method further comprises one or more of the following: inducing primary leaves from the base of its adjacent epicotyl bud formation; shoots cultured in a medium containing the selection agent; selection of transformed bud; and the transformed plant from the transformed shoot regeneration.

在进一步的实施方案中,本发明提供了生产稳定转化的大豆植物的方法,所述方法包括:(a)通过以下步骤从大豆种子制备外植体:(i)从所说的种子中去除全部或部分下胚轴;(ii)从所说的种子中去除一个子叶及其邻近的腋芽,并且保留一个其上附有上胚轴和初生叶的子叶;(iii)从上胚轴中去除部分初生叶,由此产生至少一个初生叶基部;(b)共培养该外植体和含有待掺入到大豆细胞基因组的目的核酸的农杆菌;(c)从初生叶基部区诱导芽形成;(d)在含有选择剂的培养基中培养形成的芽;(e)选择转化的芽;以及(f)再生已选择的转化芽为大豆植物。 In a further embodiment, the present invention provides a method for producing stably transformed soybean plant, the method comprising: (a) from soybean seeds explants prepared by the following steps: (i) removing from said seeds all or hypocotyl section; (ii) removing from said seed and a cotyledon adjacent axillary buds, and retained thereon with a cotyledon and epicotyl primary leaves; (iii) removing from the upper portion of the hypocotyl primary leaves, thereby generating at least one primary leaf base; (b) co-culturing the explant containing genomic be incorporated into the nucleic acid Agrobacterium soybean cells; (c) primary leaves from the base region inducing bud formation; ( d) culturing in a medium containing a selection agent bud formation; (e) selecting transformed shoots; and (f) reproducing the selected transformed shoots to soybean plants.

在另一个实施方案中,去除(a)(ii)中产生的外植体每个初生叶的一部分,从而产生一对初生叶基部。 In another embodiment, the removal of (a) (ii) a portion of the explants produced for each primary leaves to produce a pair of primary leaf base.

本发明的方法可用于将任何目的核酸导入大豆细胞中。 The method of the present invention may be used for any purpose soybean nucleic acid into a cell. 在本发明的一个实施方案中,所述核酸含有在大豆中表达所需农艺学性状的基因。 In one embodiment of the invention, the nucleic acid comprises a gene required for expression of agronomic traits in soybeans.

在本发明的另一实施方案中,所述核酸含有用作选择标记基因的磷酸甘露糖异构酶基因。 In another embodiment of the invention, the nucleic acid used as a selection marker gene comprising mannose phosphate isomerase gene.

在本发明的另一实施方案中,外植体和农杆菌共转染是在有甘露糖存在的情况下进行的。 In another embodiment of the present invention, the explant and Agrobacterium co-transfection is carried out in the presence of mannose.

成熟和未成熟种子都可用于产生本发明所用的外植体。 Mature and immature seeds can be used to produce explants used in the present invention.

附图简述图1显示质粒pNOV2105的图谱。 BRIEF DESCRIPTION Figure 1 shows a map of plasmid pNOV2105.

图2显示质粒pNOV2145的图谱。 Figure 2 shows a map of plasmid pNOV2145.

图3显示质粒pNOV2147的图谱。 Figure 3 shows a map of plasmid pNOV2147.

图4显示制备大豆外植体的示范方法。 Figure 4 shows an exemplary method for preparing the soybean explants. 图A描述了大豆种子胚胎,其中去除一部分下胚轴。 Figure A soybean seed embryonic described, wherein removing a portion of the hypocotyl. 图B描述了来自图A的大豆外植体,其中去除了一个子叶及其邻近的腋芽。 Panel B soybean explants from the map A, which removes a cotyledon and the adjacent axillary buds. 图C描述了去除两个初生叶的图B的大豆外植体,在每个初生叶的基部产生断裂点。 FIG C depict two primary leaves removed B of FIG soybean explants, the breaking point is generated in each base of the primary leaf.

图5显示质粒pBSC11234的图谱。 Figure 5 shows the map of plasmid pBSC11234.

图6显示质粒pBSC11369的图谱。 Figure 6 shows a map of plasmid pBSC11369.

发明详述现在,将参考附图在下文中更加全面地描述本发明,其中描述了本发明的多种实施方案。 DETAILED DESCRIPTION Reference will now be more fully described the present invention in the following drawings, which describe various embodiments of the present invention. 不过,本发明可以以不同的方式体现,而不应该解释为限于这里所示的实施方案。 However, the present invention may be embodied in different forms and should not be construed as limited to the embodiments shown herein. 相反,提供这些实施方案是为了使本公开更全面和完整,向本领域的技术人员全面地表达本发明的范围。 Rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the scope of the invention to those skilled in the art. 这里用于本发明描述的术语仅仅是为了说明具体的实施方案而没有限制本发明之意。 The term used herein to describe the present invention are merely to illustrate particular embodiments without limitation of the present invention is intended. 在本发明说明书和所附的权利要求中,除非文中另外清楚地指明外,单数形式“一个”和“这个”旨在也包括复数形式。 In the present invention, the specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a" and "the" are intended to include the plural forms.

除非另外定义,本文所用技术和科学术语与本发明所属领域的普通技术人员通常理解的意思相同。 Unless defined otherwise, as used herein have the same technical and scientific terms ordinary skill in the art of the present invention generally understood meaning.

除非另外说明,可使用标准方法生产根据本发明的克隆基因、表达盒子、载体(例如质粒)、蛋白和蛋白片段、以及转化的细胞和植物。 Unless otherwise specified, using standard methods cloned genes produced according to the present invention, the expression cassette, vectors (e.g. plasmids), proteins and protein fragments, as well as the transformed cells and plants. 除非另有说明,可使用标准方法生产根据本发明的克隆基因、表达盒子、载体(例如质粒)、蛋白和蛋白片段。 Unless otherwise indicated, standard methods may be used cloned genes produced according to the present invention, the expression cassette, vectors (e.g. plasmids), proteins and protein fragments. 这些技术对本领域技术人员是公知的,参见例如,J.Sambrook等,分子克隆:实验室手册第二版(冷泉港实验室,Cold Spring Habor,New York,1989),和FMAusubel等,当前分子生物学实验方案(Green PublishingAssociates,Inc.和Wiley-Interscienee,New York,1991);J.Draper等编辑,植物遗传转化和基因表达:实验室手册,(BlackwellScientific Publications,1988);和SBGelvin & RASchilperoort编辑,植物中基因产物的导入、表达和分析。 These techniques are known to those skilled in the art, see, e.g., J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory, Cold Spring Habor, New York, 1989), and the like FMAusubel current molecular biology experimental scheme (. Green PublishingAssociates, Inc, and Wiley-Interscienee, New York, 1991); J.Draper like editing, plant genetic transformation and gene expression: A laboratory Manual, (BlackwellScientific Publications, 1988); and SBGelvin & amp; RASchilperoort edit , introduced into a plant gene product, expression and analysis.

本发明描述了用目的核酸序列稳定转化大豆和再生转基因大豆植物的方法和组合物。 The present invention describes a nucleic acid sequence, stable transformation methods and compositions of soybeans and regeneration of transgenic soybean plants.

本发明的方法可用于在大豆植物中表达任何目的核酸。 The method of the present invention may be any nucleic acid of interest for expression in soybean plants. 目的基因可以是,例如,除草剂性基因、抗病基因、或抗昆虫/害虫基因,或者是选择或评价标记,并含有植物可操作的启动子、编码区和3'终止子区。 Gene of interest may be, e.g., a herbicide resistance gene, a disease resistance gene, or an anti-insect / pest gene, or a selection or evaluation of marker, and contains a plant operable promoter, coding region and 3 'terminator regions. 除草剂性基因包括对咪唑啉酮或磺酰脲除草剂耐药的AHAS基因、对bialaphos或glufosinate耐药的pat或bar基因、对glyphosate耐药的EPSP合酶基因等。 Herbicide resistance genes include imidazolinone or sulfonylurea herbicide resistant AHAS gene for resistance to bialaphos or glufosinate pat or bar gene for glyphosate resistance EPSP synthase gene and the like. 抗病基因包括抗生素合成酶基因,例如硝吡咯菌素合成酶基因,植物来源的抗性基因等等。 Antibiotic resistance genes include synthase gene, e.g. pyrrolnitrin synthase gene, a plant-derived resistance genes and the like. 昆虫抗性基因包括苏云金菌芽孢杆菌杀虫蛋白基因。 Insect resistance genes include the bacteria Bacillus thuringiensis insecticidal protein gene. 目的基因也可以编码与生物化学途径有关的酶,该酶的表达可改变食物、饲料、营养药和/或药物生产中重要的性状。 The gene may also encode related biochemical pathway, the enzyme may alter the expression of food, feed, nutraceutical and / or pharmaceutical production important trait. 目的基因可以位于质粒上。 The gene may be located on a plasmid. 适合本发明使用的质粒可以含有一个以上目的基因和/或农杆菌可含有带有不同目的基因的不同质粒。 A plasmid suitable for use according to the invention may contain more than one gene of interest and / or the Agrobacterium may comprise different plasmids having different genes of interest.

本发明提供了大豆属各种类包括Glycine max(大豆种)的转化方法。 The present invention provides a soybean belonging to different species include Glycine max (soybean seed) transformation methods. 所述方法是以农杆菌介导的目的基因传输入大豆细胞、之后转化的细胞再生为转化的大豆植物为基础的。 The method is based on Agrobacterium-mediated transfer of the gene of soybean input cells, transformed cells are then regenerated into soybean plants transformed based. 本发明的方法独立于栽培品种。 The method of the present invention is independent of the cultivars.

在本发明的一个实施方案中,外植体是通过将从温室栽培植物收集的大豆成熟种子或未成熟种子在种子发芽培养基中萌发一段时间,从所说的成熟种子或未成熟种子去除种皮,之后去除子叶而制备的。 In one embodiment of the present invention, the soybean explants are collected by the mature seeds from greenhouse plants or immature seed germination period in the seed germination medium, mature seeds from said seed or immature seeds removed skin, after removal of the cotyledons prepared. 在本发明一个优选实施方案中,然后去除一部分暴露的初生叶,从而在初生叶基部(primary leaf base)产生断裂点(图4)。 In a preferred embodiment of the present invention and then removing the exposed portion of the primary leaf to produce a breaking point (FIG. 4) in the primary leaf base (primary leaf base). 将农杆菌介导的基因传输在初生叶基部或初生叶断裂点区的细胞上进行。 Agrobacterium-mediated gene transfer is performed on the cell or the base of the primary leaf breakpoint region of the primary leaf. 从上胚轴的初生叶基部区诱导不定芽(shoots)形成。 Formation of adventitious buds (Shoots) primary leaves from the base of the hypocotyl region. 该诱导是通过去除先存在的分生组织(即初生、次生和腋生的分生组织),并将外植体在含有合适生长调节剂的芽诱导培养基中培养获得的。 This is induced by removing meristem (i.e. primary, secondary and axillary meristems) pre-existing, and explants suitable shoot induction medium containing growth regulators in the culture obtained. 所述的芽诱导方法促进了来自靶向的初生叶基部细胞的转化芽的发育或再生。 Buds method of promoting the development or regeneration of shoots from transformed primary cell targeting the leaf base.

在选择剂存在的情况下培养转化的大豆细胞。 Culturing the transformed soybean cells in the presence of a selection agent. 优选地,这些细胞用磷酸甘露糖异构酶(PMI)基因转化,并且转化的基因在甘露糖存在的情况下培养。 Preferably, these cells (PMI) gene was transformed with mannose phosphate isomerase, and culturing the transformed gene in the presence of mannose. 在含有甘露糖为选择剂的培养基中,PMI基因转化的大豆细胞比没有如此转化的大豆细胞有生长优势。 In a medium containing mannose as a selective agent, PMI gene transformed soybean cells have a growth advantage over cells not so transformed soybean.

与文献报导的其它农杆菌介导的转化方法相比,用本发明所述方法再生转化的大豆植物所需时间显著地缩短了。 The time required other Agrobacterium-mediated transformation methods reported in the literature and compared with the present invention is a method for regenerating the transformed soybean plants is significantly shortened. 从转化实验开始8到12周生根的转化大豆芽可以生成。 From transformation experiments began 8-12 weeks rooting transformed sprouts may be generated. 待插入大豆基因组中的外源遗传构建体或转基因可通过常规的重组DNA操作技术在体外构建。 Be inserted into the soybean genome exogenous genetic construct or transgene can be constructed by conventional in vitro recombinant DNA technique. 该构建体可由任何异源核酸组成。 This construct may be any heterologous nucleic acids. 该遗传构建体转化入农杆菌株中以便传输到大豆细胞中。 The genetic construct was transformed into Agrobacterium strain for transmission to the soybean cells. 所述农杆菌是非致癌的,几个这样的菌株目前可广泛地获得。 The non-oncogenic Agrobacterium Several such strains are currently widely available. 所述农杆菌优选选自根癌农杆菌(A.tumefaciens)和毛根农杆菌(A.rhizogenes)。 The Agrobacterium tumefaciens is preferably selected from (A. tumefaciens), and Agrobacterium rhizogenes (A. rhizogenes) root.

优选地,外源基因构建体含有选择标记基因。 Preferably, the exogenous gene construct containing a selectable marker gene. 优选的选择标记基因是磷酸甘露糖异构酶基因。 A preferred selection marker gene is mannose phosphate isomerase gene. 其它合适的选择标记基因包括,但不限于,编码下述物质的基因:新霉素磷酸转移酶II(Fraley等,CRCCritical Reviews in Plant Science 4,1(1986));氰氨水合酶(Maier-Greiner等,Proc.Natl.Acad.Sci.USA 88,4250(1991));天冬氨酸激酶;二氢吡啶二羧酸合酶(Perl等,BioTechnology 11,715(1993));bar基因(Toki等,Plant Physiol.100,1503(1992);Meagher等,Crop Sci.36,1367(1996));色氨酸脱羧酶(Goddijn等,Plant Mol.Biol.22,907(1993));新霉素磷酸转移酶(NEO;Southern等,J.Mol.Appl Gen.1,327(1982));潮霉素磷酸转移酶(HPT或HYG;Shimizu等,Mol.Cell.Biol.6,1074(1986));二氢叶酸还原酶(DHFR);膦丝菌素乙酰转移酶(DeBlock等,EMBO J.6,2513(1987));2,2-二氯丙酸脱卤素酶(Buchanan-Wollatron等,J.Cell.Biochem.13D,330(1989));乙酰羟基酸合酶(Anderson等,美国专利号No.4,761,373;Haughn等,Mol.Gen.Genet.221,266(1988));5-烯醇丙酮酰-莽草酸-磷酸(5-eno Other suitable selectable marker genes include, but are not limited to, genes encoding the following substances: neomycin phosphotransferase II (Fraley et, CRCCritical Reviews in Plant Science 4,1 (1986)); cyanide ammonia synthase (Maier- Greiner, etc., Proc.Natl.Acad.Sci.USA 88,4250 (1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et, BioTechnology 11,715 (1993)); bar gene ( Toki and so on, Plant Physiol.100,1503 (1992); Meagher, etc., Crop Sci.36,1367 (1996)); tryptophan decarboxylase (Goddijn, etc., Plant Mol.Biol.22,907 (1993)); new neomycin phosphotransferase (NEO; Southern et, J.Mol.Appl Gen.1,327 (1982)); hygromycin phosphotransferase (HPT or HYG; Shimizu et, Mol.Cell.Biol.6,1074 ( 1986)); dihydrofolate reductase (the DHFR); phosphinothricin acetyltransferase (DeBlock et, EMBO J.6,2513 (1987)); 2,2- dichloropropionic acid dehalogenase (Buchanan-Wollatron etc., J.Cell.Biochem.13D, 330 (1989)); acetohydroxyacid synthase (Anderson et al., U.S. Patent No. No.4,761,373; Haughn et, Mol.Gen.Genet.221,266 (1988)); 5 - enolpyruvyl acid - shikimate - phosphate (5-eno lpyruvyl-shikimate-phosphate)合酶(aroA;Comai等,Nature 317,741(1985));卤芳基腈水解酶(Stalker等,WO87/04181);乙酰辅酶A羧化酶(Parker等,Plant Physiol.92,1220(1990));二氢蝶酸合酶(sul I;Guerineau等,Plant Mol.Biol.15,127(1990));以及32kDa的光系统II多肽(psbA;Hirschberg等,Science 222,1346(1983))。 lpyruvyl-shikimate-phosphate) synthase (aroA; Comai et, Nature 317,741 (1985)); haloaryl nitrilase (Stalker et, WO87 / 04181); acetyl coenzyme A carboxylase (Parker et, Plant Physiol .92,1220 (1990)); dihydropteroate synthase (sul I; Guerineau et, Plant Mol.Biol.15,127 (1990)); and the 32kDa photosystem II polypeptide (psbA; Hirschberg et, Science 222 , 1346 (1983)).

还包括的基因有编码对以下药物耐药的基因:氯霉素(Herrera-Estrell等,EMBO J.2,987(1983));氨甲蝶呤(Herrera-Estrella等,Nature 303,209(1983);Meijer等,PlantMol.Biol.16,807(1991));潮霉素(Waldron等,Plant Mol.Biol.5,103(1985);Zhijian等,Plant Science 108,219(1995);Meijer等,Plant Mol.Bio.16,807(1991));链霉素(Jones等,Mol.Gen.Genet.210,86(1987));壮观霉素(Bretagne-Sagnard等,TransgenicRes.5,131(1996));博来霉素(Hille等,Plant Mol.Biol.7,171(1986));磺胺(Guerineau等,Plant Mol.Bio.15,127(1990));bromoxynil(Stalker等,Science 242,419(1988));2,4-D(Streber等,Bio/Technology 7,811(1989));膦丝菌素(DeBlock等,EMBOJ.6,2513(1987));壮观霉素(Bretagne-Sagnard和Chupeau,Transgenic Research 5,131(1996))。 Further comprising a gene encoding the drug resistance gene: chloramphenicol (Herrera-Estrell the like, EMBO J.2,987 (1983)); methotrexate (Herrera-Estrella et, Nature 303,209 (1983 ); Meijer et, PlantMol.Biol.16,807 (1991)); hygromycin (Waldron et, Plant Mol.Biol.5,103 (1985); Zhijian et, Plant Science 108,219 (1995); Meijer et , Plant Mol.Bio.16,807 (1991)); streptomycin (Jones et al, Mol.Gen.Genet.210,86 (1987)); spectinomycin (Bretagne-Sagnard et, TransgenicRes.5,131 ( 1996)); bleomycin (Hille et, Plant Mol.Biol.7,171 (1986)); sulfonamide (Guerineau et, Plant Mol.Bio.15,127 (1990)); bromoxynil (Stalker et, Science 242 , 419 (1988)); 2,4-D (Streber et, Bio / Technology 7,811 (1989)); phosphinothricin (DeBlock et, EMBOJ.6,2513 (1987)); spectinomycin (Bretagne -Sagnard and Chupeau, Transgenic Research 5,131 (1996)).

在一个实施方案中,用于转化方法的起始材料是大豆成熟种子。 In one embodiment, the starting material for the conversion process is mature seeds of soybean. 在另一个实施方案中,起始材料可以是栽培大豆植物的大豆未成熟种子。 In another embodiment, the starting material may be cultivated soybean immature seeds of a soybean plant. 所述种子置于发芽培养基中,允许其发芽6-24小时,优选地发芽大约6-14小时,更优选地大约8-12小时。 The seed germination medium was placed and allowed to germinate for 6-24 hours, preferably about 6-14 hr germinated, more preferably about 8-12 hours. 如果需要的话,还可以允许种子发芽更长时间,例如2到5天。 If desired, seed germination may also allow a longer time, for example 2-5 days.

去除发芽种子的种皮和下胚轴。 Remove the seed coat and hypocotyls germinated seeds. 再去除一个子叶及其邻近的腋芽。 Then remove one cotyledon and the adjacent axillary buds. 然后,将初生叶实质性地去除,从而产生一个含有初生叶基部、该初生叶基部附着的上胚轴以及该上胚轴附着的子叶的外植体。 Then, substantially the primary leaves removed, thereby producing a primary blade comprising a base, the base portion adhered to the primary leaves and hypocotyl explants on the hypocotyl cotyledon attached. 实质性地去除意思是去除了初生叶组织的主要部分。 Substantive meaning to remove the main part of the primary leaf tissue removed.

对于农杆菌介导的基因转移,已知植物组织的创伤能促进基因转移。 For Agrobacterium-mediated gene transfer, trauma known plant tissue can promote gene transfer. 因此,优选但不是必需的,可在初生叶基部区域造成伤口。 Thus, preferably, but not necessarily, the wound may cause the primary leaf base area.

然后,将如上文所述方法制备的外植体浸入农杆菌细胞悬浮液中几分钟到几小时,典型地大约0.5-3小时,优选地1-2小时。 Then, the explants were prepared as described above was immersed in the Agrobacterium cell suspension method in minutes to hours, typically about 0.5-3 hours, preferably 1-2 hours. 去除过多的农杆菌细胞悬浮液,允许剩下的农杆菌和外植体在大约22℃±2℃的温度、16小时光照/8小时黑暗条件下在共培养培养基上共培养几天,典型地2到5天,优选地3到4天。 Removing excess Agrobacterium cell suspension, allowing the rest of the explant and Agrobacterium at about 22 ℃ ± 2 ℃ temperature, 16 h light / 8 h dark conditions in the co-culture media on days co-cultivation, typically 2-5 days, preferably 3-4 days.

共培养后,将外植体转移到一个培养基(或一系列培养基)中培养8-12周,所述培养基有利于芽发育和转化细胞的选择。 After co-cultivation, explants are transferred to a medium (or a series of medium) culturing 8-12 weeks, shoots develop the culture medium and facilitate selection of transformed cells. 这样的培养基(或这些培养基)一般含有芽诱导激素以及选择剂。 Such media (or the media) typically contain a selection agent hormones and shoot induction. 下面实施例中使用的再生培养基含有甘露糖作为选择剂,以及苄氨基嘌呤作为芽诱导激素。 Used in the following Examples regeneration medium containing mannose as an embodiment of a selective agent, as well as benzylaminopurine buds hormone. 术语激素还包括诱导芽形成的细胞生长调节化合物,包括但不限于植物生长素(例如IAA、NAA和吲哚丁酸(IBA))、细胞分裂素(cytokinins)(如thidiazuron、激动素(kinetin)和玉米素),和/或赤霉素(GA3)。 The term hormone-induced shoot formation further comprises a cell growth regulating compounds, including but not limited to auxins (e.g. IAA, NAA and indole butyric acid (IBA)), cytokinins (Cytokinins) (eg Thidiazuron, kinetin (Kinetin) and zeatin), and / or gibberellin (GA3).

当芽长到大约2cm并有完全三叶形成时,将芽从外植体分出,并置于生根培养基上诱导根的形成。 When the bud to fully clover about 2cm and have formed the shoots separated from the explants and placed on rooting formed root induction medium. 优选地,生根培养基也包括选择剂以进一步有助于鉴定可能转化的芽。 Preferably, the rooting medium comprising a selection agent also helps to further identify potential transformed shoots. 根形成大约需要1-2周,之后可将该植物转移到土壤中并培养到完全成熟。 Root formation takes about 1-2 weeks, the plants can then be transferred to soil and grown to full maturity.

含有异源核酸(即含有按照本文描述方法转化的细胞或组织)的转基因植物以及通过该转基因植物产生的种子和后代是本发明的另一方面。 Containing a heterologous nucleic acid (i.e., a cell or tissue comprising transformed according to the method described herein) and by a transgenic plant seeds and progeny of the transgenic plants is another aspect of the present invention. 将转化细胞培养成有用栽培品种的方法是本领域技术人员公知的。 The transformed cells into a useful method cultivars are known to those skilled in the art. 植物组织体外培养技术和许多情况中整体植物再生技术是公知的。 Plant tissue culture in vitro and in many cases the whole plant regeneration techniques are well known. 本发明的另一方面是含有上述核酸的转基因植物组织、植物或种子。 Another aspect of the present invention is a transgenic plant tissue, plants or seeds containing the nucleic acid. 在优选的实施方案中,使用本文所述方法产生的转化植物不是嵌合的,或仅仅有一小部分转化的植物是嵌合的。 In a preferred embodiment, it generated using the methods described herein are not chimeric transformed plant, or only a small portion of the chimeric transformed plant. 优选地,这通过延长高浓度细胞分裂素处理的时间或增加甘露糖选择的严紧性或两者皆用而实现。 Preferably, this time is extended by a high concentration of cytokinin treatment or increase the stringency of selection of mannose or both be implemented.

因此,通过选择或筛选鉴定并在如本文所提供的支持再生的合适培养基中培养后,可以允许本发明的转化细胞成熟为植物。 Thus, after culturing in a suitable medium and support as provided herein regenerated identified by selection or screening, transformed cells may be allowed to mature plants according to the present invention. 优选地,植物在栽培间或者温室中成熟。 Preferably, between the cultivated plant maturation or greenhouse. 转化子鉴定后大约2-6周(依赖于初始的组织),植物得以再生。 Transformants were identified after about 2-6 weeks (depending on the initial tissue), the plant is regenerated. 在再生期间,细胞可在组织培养容器中的固体培养基上培养。 During regeneration, cells may be cultured in tissue culture on a solid medium container. 这些培养容器的示例实施方案是培养皿和植物Cons。 The exemplary embodiment of the plate and the culture container are plants Cons. 再生中的植物长到芽和根发育阶段后,为进一步生长和试验可以将它们转移到温室中。 After the plant reached the stage of shoot and root development, reproduction, and for the further growth and testing, they can be transferred to the greenhouse. 如上文所提供的,再生植物的种子和后代植物是本发明的一个方面。 As provided above, the regeneration of plants seeds and progeny plants are an aspect of the present invention. 相应地,术语“种子”意指包括这些转化植物的种子以及转化植物后代产生的种子。 Accordingly, the term "seed" is meant to include progeny of transformed plants Seeds and these generated transformed plants. 本发明的植物不仅包括转化和再生的植物,还包括通过本文所述方法产生的转化和再生植物的后代。 Plants of the present invention include not only the plant transformation and regeneration, and further comprising transforming the progeny of the regenerated plants produced by the methods described herein.

通过上文所描述的标准方法可以从所述方法产生的植物中筛选成功的转化植物。 The method can be screened from the plant produced by standard methods described above successfully transformed plants. 为了开发改良的植物和种子品系(这也是本发明的另一方面),可以持续地筛选和选择本发明再生植物的种子和子代植物以使转基因和整合的核酸序列持续存在。 , The screening and selection may be continuously regenerated plants as the present invention and progeny transgenic plants and integration of nucleic acid sequences in order to develop improved plant and seed lines (this is also another aspect of the present invention) persists. 因此,可以将所需要的转基因核酸序列移入(即渐渗或近亲交配)其它的遗传品系如某些原种或商业上有用的品系或品种中。 Thus, the nucleic acid sequence desired transgene may be moved (i.e. introgression or inbred) lines as other genetic elite some useful or commercial line or variety. 渐渗目的核苷酸序列进入遗传植物品系的方法可通过本领域公知的多种技术来实现,这些技术包括通过传统的育种、原生质体融合、细胞核转移以及染色体转移。 The method of introgression nucleotide sequence into the plant genetic strains may be achieved by a variety of well known in the art of techniques including through classical breeding, protoplast fusion, nuclear transfer and chromosome transfer. 育种方法和技术是本领域公知的,例如下列文献中所示的,JRWelsh,植物遗传和育种基础(John Wiley和Sons,New York,(1981));农作物育种(DRWood,编辑,American Society of Agronomy,Madison,Wisconsin,(1983));O.Mayo,植物育种理论,第二版(ClarendonPress,Oxford,England(1987);以及Wricke和Weber,数量遗传学和选择植物育种(Walter de Gruyter和Co.,Berlin(1986))。使用这些以及本领域的其它技术,根据本发明获得的转基因植物和近交系可用于生产商业上有价值的杂交植物和农作物,杂交的品种也是本发明的一个方面。 Breeding methods and techniques are known in the art, e.g., JRWelsh, plant genetics and breeding base shown in the following literature (John Wiley and Sons, New York, (1981)); plant breeding (DRWood, Editor, American Society of Agronomy , Madison, Wisconsin, (1983)); O.Mayo, plant breeding theory, second Edition (ClarendonPress, Oxford, England (1987); and Wricke and Weber, quantitative genetics and plant breeding selection (Walter de Gruyter and Co. , Berlin (1986)). using these and other techniques in the art, the transgenic plants according to the present invention is to obtain hybrid plants and crops, hybrid varieties of valuable production business and inbred lines may be used is the present invention to an aspect.

上述内容是本发明各种实施方案的举例说明,而不应看作是对其限制。 The foregoing is illustrative of the various embodiments of the invention, and should not be regarded as limiting thereof.

通过下面的实施例进一步描述本发明,这些实施例并不以任何方式限制本发明的范围。 The present invention is further described by the following examples, these examples do not limit the scope of the invention in any manner.

实施例1转化载体质粒pNOV2105(图1)是对WO 97/04112公开和描述的pVictor的修改,其中35S启动子由SMAS启动子代替,35S终止子由Nos终止子代替,另一SMAS启动子插入到GUS内含子GUS序列的上游,GUS内含子GUS序列3'末端侧翼是Nos终止子。 Example 1 Plasmid transformation vector pNOV2105 (FIG. 1) is disclosed embodiment and modifications described pVictor WO 97/04112, wherein a 35S promoter replaced SMAS promoter, a 35S terminator Nos terminator in place, another promoter inserted SMAS upstream intron GUS GUS sequence, GUS GUS intron sequences 3 'end flanked Nos terminator. 用于本文所述方法中的pNOV2105不含有在pVictor中发现的多克隆位点。 pNOV2105 used in the methods described herein do not contain a multiple cloning site found in the pVictor. 不过,如果需要的话,增加这样的克隆位点完全在本领域的技术之内。 However, if desired, this increase in fully cloning site of the skill of the art.

pNOV2105(图1)是用于农杆菌介导的植物转化的载体,并含有来源于胭脂氨酸型pTiT37质粒(Yadav等,1982 Proc Natl Acad Sci79:6322-6326)的Ti右和左边界序列,其位于磷酸甘露糖异构酶(PMI)和β-葡糖醛酸酶(GUS)基因的侧翼。 pNOV2105 (FIG. 1) is a vector for Agrobacterium-mediated plant transformation, and containing from nopaline type plasmid pTiT37 (Yadav et, 1982 Proc Natl Acad Sci79: 6322-6326) right and left border sequence of the Ti, located phosphomannose isomerase (PMI) and flanking β- glucuronidase (GUS) gene.

为在大肠杆菌(E.coli)中复制和保持,该质粒含有大肠杆菌质粒pUC19的复制原点(pUC19ori)(Yanish-Perron等,1985 Gene33:103-119),并且为了在根癌农杆菌中复制和保持,该质粒还含有假单胞菌(Pseudomonas)质粒pVS1的复制原点(pVS1ori)(Itoh等1984 Plasmid 11:206-220;Itoh和Hass 1985 Gene 36:27-36)。 And held for replication in E. (E.coli), E. coli plasmid pUC19 plasmid contains the origin of replication (pUC19ori) (Yanish-Perron et, 1985 Gene33: 103-119), and to replicate in Agrobacterium tumefaciens and holding, the plasmid further contains Pseudomonas (of Pseudomonas) plasmid pVS1 origin of replication (pVS1ori) (Itoh et 1984 plasmid 11: 206-220; Itoh and Hass 1985 Gene 36: 27-36). 为在大肠杆菌和根癌农杆菌中筛选,该质粒含有编码3″(9)-O-核苷酸转移酶的来自转座子Tn7的壮观霉素/链霉素抗性基因(spec/strep)(Fling等1985 Nucleic Acids Res 19:7095-7106)。为在细菌中有效的表达,spec/strep抗性基因与tac启动子(参见如Amann等1983 Gene 25(203):167-78)融合。 Screening of E. coli and Agrobacterium tumefaciens, a plasmid containing the coding 3 "(9) -O- deoxynucleotidyl transferase spectinomycin derived from the transposon Tn7 / streptomycin resistance gene (spec / strep ) (Fling et 1985 Nucleic Acids Res 19: 7095-7106) for the effective expression in bacteria, spec / strep resistance gene and the tac promoter (see, e.g., Amann et al. 1983 gene 25 (203.): 167-78) fusion .

在右和左边界之间的T-DNA片段含有下述基因,这些基因是通过根癌农杆菌介导的转化转移到大豆植物中的仅有基因。 T-DNA between the right and left border fragment containing the genes, these genes are transferred into the soybean plants only gene by transformation Agrobacterium mediated cancer.

GUS内含子GUSβ-葡糖醛酸酶(GUS):该片段挨着右边界,含有大肠杆菌的β-葡糖醛酸酶基因,在该编码区内有一内含子以阻止融合SMAS启动子和Nos终止子的农杆菌进行翻译。 GUS intron GUSβ- glucuronidase (GUS): next to the right border fragment, containing the β- glucuronidase gene of E. coli, a fusion SMAS to prevent intron promoter the coding region Agrobacterium and Nos terminator for translation. GUS内含子GUS基因是从质粒pBISN1(Narasimhulu等1986烟草和玉米中农杆菌DNA的早期转录,PlantCell 8:873-866)分离的。 GUS intron GUS gene from plasmid pBISN1 (1986 tobacco and maize DNA in Agrobacterium Narasimhulu other early transcription, PlantCell 8: 873-866) isolated.

磷酸甘露糖异构酶(PMI):该片段挨着左边界,是来自大肠杆菌的甘露糖-6-磷酸异构酶基因(Miles和Guest 1984,Gene 32:41-48),与SMAS启动子(NiM,Cui D,Einstein J,NarasimhuluS,Vergara CE,Gelvin SB(1995))和Nos终止子融合。 Mannose phosphate isomerase (PMI): next to the left border fragment, mannose-6-phosphate isomerase gene from E. coli (Miles and Guest 1984, Gene 32: 41-48), and SMAS promoter (NiM, Cui D, Einstein J, NarasimhuluS, Vergara CE, Gelvin SB (1995)) and Nos terminator fusion. 磷酸甘露糖异构酶基因作为选择标记以在含有D-甘露糖为碳源的培养基上筛选转基因芽。 Mannose phosphate isomerase gene as a selection marker to D- mannose containing screening transgenic shoots on a medium as a carbon source.

pNOV2145(图2)的组成和序列如SEQ ID NO:1所示。 pNOV2145 (FIG. 2) composition and sequence as SEQ ID NO: 1 shown in FIG. pNOV2147(图3)的组成和序列如SEQ ID NO:2所示。 pNOV2147 (FIG. 3) of the composition and sequence as SEQ ID NO: 2 shown in FIG.

实施例2转化和再生通过在干燥器中释放氯气对成熟干燥的大豆种子(Var.S42 H1)进行表面消毒。 Example 2 Transformation and regeneration by releasing chlorine gas in a drier for drying the mature soybean seeds (Var.S42 H1) are surface sterilized. 种子保存在petri培养皿中,氯气通过在烧杯中倒入100ml Clorox并慢慢加入8ml浓HCL产生。 Seed is stored in petri dish, was poured into 100ml Clorox by chlorine in a beaker and slowly added to 8ml of concentrated HCL generated. 种子至少用两次氯气释放处理来消毒,每次持续8-18小时。 Seeds released at least twice with chlorine disinfection treatment, each lasting 8-18 hours.

然后,将消毒的种子(每皿大约15-20个种子)置于含有0.6%琼脂凝固的MS基础培养基(Murashige和Skoog(1962)烟草愈伤组织培养物快速生长和生化鉴定的修改培养基。Physiol Plant 15:473-479)和2%蔗糖的发芽培养基上。 Then, the sterilized seeds (about 15-20 seeds per dish) was placed modified medium containing 0.6% agar solidified MS basal medium (Murashige and Skoog (1962) was rapid growth of tobacco callus culture and biochemical identification .Physiol Plant 15: 473-479) and 2% sucrose on germination medium. pH值保持5.8。 pH is maintained 5.8. petri培养皿置于37℃房间使种子过夜生长或吸涨。 petri dish, at 37 ℃ grown overnight or room so that imbibed seeds. 去除种皮,然后去除部分下胚轴,保留大约0.5cm的下胚轴。 Removing the seed coat, and then removing a portion of the hypocotyls, about 0.5cm hypocotyl of retention. 去除一个子叶连同其邻近的腋芽并丢弃。 Together with the removal of one adjacent axillary cotyledons and discarded. 在剩下的子叶上,用手术刀断开初生叶,保留上胚轴上的初生叶基部(图4)。 On the remaining cotyledons, primary leaves is disconnected with a scalpel, retained on the primary leaf base the epicotyl (FIG. 4).

将冻存的甘油储备液中保存的含有质粒pNOV2145(ZsGreen1和PMI,如实施例1所述)的农杆菌菌株(LBA 4404)划线在含有合适抗生素(100mg/L壮观霉素)的YEP(酵母提取物10g/L,蛋白胨5g/L,氯化钠5g/L,细菌培养用琼脂15g/L)培养皿上。 The glycerol stock was stored frozen Agrobacterium strain (LBA 4404) containing the plasmid pNOV2145 (ZsGreen1 and PMI, as described in Example 1) in the scribe YEP containing appropriate antibiotics (100mg / L spectinomycin) of ( yeast extract 10g / L, peptone 5g / L, NaCl 5g / L, with a bacterial culture on agar 15g / L) Petri dishes. 然后,将农杆菌27℃温育1-2天。 Then, 27 ℃ Agrobacterium incubated for 1-2 days. 从培养皿上取一勺农杆菌放入100ml含有抗生素(100mg/L壮观霉素)的YEP液体培养基中在摇床上27℃培养过夜。 Take a spoonful of YEP liquid medium from the culture dishes into Agrobacterium 100ml antibiotics (100mg / L spectinomycin) shaker overnight culture 27 ℃. 细菌悬液在大约1500g离心15分钟,然后重悬在共培养液体培养基中达密度OD660=0.2或0.65,该培养基的组成为:B5盐0.05×(Sigma)、B5维生素(0.05×)(B5维生素组合物(1×):肌醇100mg/L、烟酸1mg/L、盐酸吡哆醇1mg/L、盐酸硫胺素10mg/L)、乙酰丁香酮40mg/L、蔗糖20g/L、BAP 2mg/L、GA30.25mg/L、MES(吗啉代乙烷磺酸)3.9g/L及pH5.4。 Bacterial suspension was centrifuged at about 1500g 15 minutes and then resuspended in liquid co-cultivation medium density reached OD660 = 0.2 or 0.65, the composition of culture medium: B5 salt 0.05 × (Sigma), B5 vitamins (0.05 ×) ( B5 vitamins composition (1 ×): myo-inositol 100mg / L, nicotinic acid 1mg / L, pyridoxine hydrochloride 1mg / L, thiamine hydrochloride 10mg / L), acetosyringone 40mg / L, sucrose 20g / L, BAP 2mg / L, GA30.25mg / L, MES (morpholino ethane sulfonic acid) 3.9g / L and pH5.4.

将含有靶组织的外植体浸入农杆菌悬浮液中,并孵育1-2小时。 The explants containing the target tissue is immersed in the Agrobacterium suspension and incubated for 1-2 hours. 倒掉农杆菌悬液,然后将处理的外植体置于共培养培养皿中的滤纸上。 Agrobacterium suspension drained, then treated explants were cultured on petri dishes were placed in filter paper. 保持该外植体的近轴侧和滤纸接触。 Holding the adaxial side in contact with the filter paper and the explants. 共培养固体培养基由B5盐(Sigma,0.05×)、B5维生素(0.05×)、40mg/L乙酰丁香酮、蔗糖20g/L、BAP 2mg/L、GA30.25mg/L、MES 3.9g/L及pH5.4组成。 The solid co-cultivation medium consisting of B5 salts (Sigma, 0.05 ×), B5 vitamins (0.05 ×), 40mg / L acetosyringone, sucrose 20g / L, BAP 2mg / L, GA30.25mg / L, MES 3.9g / L and pH5.4 components. 该培养基用0.5%精制琼脂(Sigma)固化。 The medium was solidified with 0.5% purified agar (Sigma).

将外植体和农杆菌在16小时光照/8小时黑暗条件下在20-30℃共培养2-5天。 The explants and Agrobacterium at 16 h light / 8 h dark conditions coculture 20-30 ℃ 2-5 days. 共培养后,在含有250mg/L氨噻肟头孢霉素的无菌水中漂洗外植体,去除初生和次生分生组织,然后将外植体转移到再生培养基(即REG-1培养基)中。 After co-culture, in sterile water containing 250mg / L cefotaxime to cefotaxime explants rinsing, removing primary and secondary meristem and the explants are transferred to regeneration medium (i.e., medium REG-1 )in. 在再生过程中,还去除邻近子叶的腋生芽以促进初生叶基部区的生长。 During regeneration, axillary buds is also removed adjacent the cotyledon to promote growth of the base region of the primary leaf.

REG-1培养基含有MS盐(1×)、B5维生素(1×)、KNO31g/L、BAP 1mg/L、替卡西林(ticarcillin)300mg/L、氨噻肟头孢霉素100mg/L、谷氨酰胺250mg/L、天冬酰胺50mg/L、甘露糖15-30g/L、蔗糖0、0.25和1g/L、pH5.6以及精制琼脂10g/L。 REG-1 medium containing MS salts (1 ×), B5 vitamins (1 ×), KNO31g / L, BAP 1mg / L, ticarcillin (ticarcillin) 300mg / L, cefotaxime cefotaxime 100mg / L, Valley amide 250mg / L, asparagine 50mg / L, mannose 15-30g / L, sucrose, 0, 0.25 and 1g / L, pH5.6 and purified agar 10g / L. 在每个petri培养皿中以直立姿势放置五个外植体,以便外植体的上胚轴末端插入培养基中。 Placed in an upright posture five explants per petri dish, so that the upper end of the hypocotyl explants inserted medium. 将培养皿置于塑料容器内,并放在22-25℃的培养间里,在每18-20小时光照/4-6小时黑暗的周期、光强度60-100μEm-2S-1的条件下培养。 The dishes were placed in a plastic container, and placed between 22-25 deg.] C is cultured, in the dark cycle every 18-20 hours light / 4-6 hours, cultured under conditions of light intensity of 60-100μEm-2S-1 . 在REG-1培养基上生长2周后,将外植体转移到REG-2培养基上,REG-2培养基含有MS盐(1×)以及B5维生素(1×)、KNO31g/L、BAP 0.5mg/L、替卡西林300mg/L、氨噻肟头孢霉素100mg/L、谷氨酰胺250mg/L、天冬酰胺50mg/L、甘露糖15g/L和蔗糖1g/L。 On the REG-1 After 2 weeks of growth medium, the explants were transferred to medium REG-2, REG-2 medium containing MS salts (1 ×), and B5 vitamins (1 ×), KNO31g / L, BAP 0.5mg / L, ticarcillin 300mg / L, cefotaxime cefotaxime 100mg / L, glutamine 250mg / L, asparagine 50mg / L, mannose 15g / L sucrose and 1g / L. 培养基的pH值保持在5.6,培养基用精制琼脂10g/L固化。 pH of the medium is maintained at 5.6, the medium was purified by agar 10g / L cure.

在4-6周,将大豆培养物转移到REG-3培养基上继续选择和芽形成。 In 4-6 weeks, the culture was transferred to a soybean REG-3 medium and selection continued bud formation. REG-3培养基含有MS盐(1×)、B5维生素(1×)、KNO31g/L、BAP 0.2mg/L、GA30.5mg/L、IBA 0.1mg/L、替卡西林300mg/L、氨噻肟头孢霉素100mg/L、谷氨酰胺250mg/L、天冬酰胺50mg/L、甘露糖15g/L、蔗糖1g/L,pH5.6,培养基用精制琼脂10g/L固化。 REG-3 medium containing MS salts (1 ×), B5 vitamins (1 ×), KNO31g / L, BAP 0.2mg / L, GA30.5mg / L, IBA 0.1mg / L, ticarcillin 300mg / L, ammonia cefotaxime, cefotaxime 100mg / L, glutamine 250mg / L, asparagine 50mg / L, mannose 15g / L, sucrose 1g / L, pH5.6, purified agar medium was 10g / L cure. 去除死的组织,带有再生芽的外植体每两周用新鲜REG-3培养基传代培养。 Removing dead tissue, with regeneration bud explants biweekly REG-3 medium with fresh subculture. 当芽长到大约2-4cm长的时候,连续从培养物上收获伸长的芽。 When the shoot to about 2-4cm long time, the continuous elongated shoots harvested from the culture. 同时,将芽转移到生根培养基中,生根培养基含有MS盐(0.5×)、B5维生素(0.5×)、谷氨酰胺250mg/L、天冬酰胺50mg/L、KNO31g/L、氨噻肟头孢霉素100mg/L、替卡西林300mg/L、蔗糖15g/L、IBA 0.5mg/L,pH5.6以及精制琼脂10g/L。 At the same time, the shoots transferred to rooting medium in the rooting medium containing MS salts (0.5 ×), B5 vitamins (0.5 ×), Glutamine 250mg / L, asparagine 50mg / L, KNO31g / L, cefotaxime cefotaxime 100mg / L, ticarcillin 300mg / L, sucrose 15g / L, IBA 0.5mg / L, pH5.6 and purified agar 10g / L.

将表达荧光蛋白基因(ZsGreen1)的生根转基因芽转移到含有湿润的Fafard发芽混合物(Conrad Fadard Inc.,MA,USA)的211培养罐中,并用塑料杯覆盖保湿继续培养大约2周。 Rooting expressing fluorescent protein gene (ZsGreen1) Transgenic shoots were transferred to moistened germination mixture containing Fafard (Conrad Fadard Inc., MA, USA) 211 culture tank, a plastic cup and covered with a moisture cultured for about 2 weeks. 植物在白天温度27-29℃、夜间温度21℃、16小时光照周期(光强度20-40μEm-2S-1)的条件下驯化。 Plant acclimation temperature during the day 27-29 ℃, night temperature 21 ℃, under 16 hr light cycle (light intensity 20-40μEm-2S-1) a. 当新叶开始长出,将植物转入含有土壤混合物的一加仑培养罐中,该土壤混合物由50-55%松树树皮堆肥、40-45%泥炭、5-10%珍珠岩(Sungrow Horticultural Supply,Pine Bluff,Arkansas)组成。 As the leaves started to grow, the plants were transferred to a culture tank containing one gallon of the soil mixture, the soil mixture of 50-55% pine bark compost, peat 40-45%, 5-10% perlite (Sungrow Horticultural Supply , Pine Bluff, Arkansas) components. 驯化的大豆植物在温室中白天温度27-29℃、夜间温度21℃、光强度400-600μEm-2S-1、相对湿度70-95%和16小时光照周期的条件下生长。 Domesticated soybean plants in the greenhouse during the day temperature 27-29 ℃, night temperature 21 ℃, light intensity 400-600μEm-2S-1, relative to the lower 16 hours photoperiod and humidity of 70-95% growth. 这些植物在生长期间用osmocote(Scotts-SierraHorticultural Products Company,Ohio;17-6-12)施肥两次(5-8g/加仑土壤)。 These plants use osmocote (Scotts-SierraHorticultural Products Company, Ohio; 17-6-12) twice during the growing fertilization (5-8g / gallon soil). 通过Taqman分析在温室栽培植物叶子中有荧光蛋白基因以及PMI基因的存在证实有转化发生。 By Taqman analysis and the presence of the gene of the fluorescent protein gene PMI confirmed conversion occurs in the leaves of plants in the greenhouse. 通过使用荧光显微镜显现表达也证实了在转化的大豆组织中荧光蛋白基因的表达。 Visualized by using a fluorescent microscope also confirmed the expression of fluorescent protein gene expression in transformed soybean tissue.

通过Southern印迹分析证实了用基因构建体pNOV2145获得了六个转基因植物。 It was confirmed by Southern blot analysis with a gene construct obtained six pNOV2145 transgenic plants. 对一个事件的后代分析PMI基因或ZsGreen1基因揭示在转化大豆的基因组中有一个T-DNA整合位点。 Analysis of progeny of a PMI event ZsGreen1 gene or genes reveals a T-DNA integration site in the genome of the transformed soybeans. 该子代在T1代中以3:1比例分离。 The T1 generation progeny in 3: 1 ratio of separation.

表1 转化的表达荧光蛋白基因(ZsGreen1)的芽 Table 1 bud transformed with the expression of fluorescent protein gene (ZsGreen1) of

实施例3将大豆种子(Var.S42 H1)表面消毒,并如实施例2所述制备外植体。 3 Soybean seeds (Var.S42 H1) EXAMPLE surface sterilized, and was prepared as described in Example 2 explant.

如实施例1所述制备含有质粒pNOV2147的农杆菌菌株(LBA4404)。 The Agrobacterium strain (LBA4404) Preparation of a plasmid containing pNOV2147 described in Example 1. 用共培养液体培养基将细菌终浓度调整为OD660=0.60。 Co-culture broth with a final bacterial concentration was adjusted to OD660 = 0.60. 外植体制备、农杆菌接种以及共培养的条件与实施例2所述的相同。 Same explant preparation, Agrobacterium inoculation and co-culture conditions described in Example 2.

在固体共培养培养基中共培养三天之后,将过多的农杆菌漂洗掉,去除初生和次生分生组织,并将外植体转移到REG-1培养基中。 After the solid co-cultivation medium co-cultured for three days, the excess Agrobacterium rinsed to remove primary and secondary meristem and the explants are transferred to medium of the REG-1. 将它们在28-30℃16小时光/8小时黑暗条件下培养。 They were incubated at 28-30 ℃ 16 hour light / 8 h dark conditions. 在REG-1培养基上培养2周后,将培养物转移到REG-2培养基中。 After 2 weeks of culture on medium REG-1, the culture was transferred to REG-2 medium. 在该再生过程中,仅保留从初生叶基部生出的芽。 In this regeneration process, leaving only the base of the primary leaf buds birth. 在大约第四周,将芽培养物转移到REG-3培养基中。 At about fourth week, the culture was transferred to shoot REG-3 medium. 然后,每10-14天转移到新鲜的REG-3培养基中。 Then, transferred to fresh medium REG-3 every 10-14 days. 如在REG-1和REG-2培养基中一样,仅保留从初生叶基部产生的芽而去除其余芽。 As in the REG-1 REG-2 medium and leaving only shoots produced from the base to remove the remaining primary leaf buds. 当延伸的芽长度达到约2-4cm时,将它们与芽培养物的其它部分分开,并转移到生根培养基中。 When the shoots reached about 2-4cm length extension, which will be separated from the rest of the shoot cultures and transferred to rooting medium.

从35个外植体中鉴定出五个表达蓝荧光蛋白基因的转基因芽(表2)。 35 identified from explants transgenic shoots five blue fluorescent protein gene expression (Table 2).

表2 转化的表达蓝荧光蛋白(cyano fluorescent protein)基因的芽。 Table 2 transformed shoots expressing blue fluorescent protein (cyano fluorescent protein) gene.

实施例4共培养期间的甘露糖处理在本实施例中所用的基因构建体是pNOV2145(其含有ZsGreen1和PMI基因,如实施例1中所述)。 Gene used in the examples mannose construct during culture in Example 4 were treated in the present embodiment is pNOV2145 (PMI genes and containing ZsGreen1, as described in Example 1). 制备外植体、农杆菌悬液以及细菌悬液接种外植体的方法如实施例2中所述的进行。 The method of preparation of the explants, and the suspension of Agrobacterium bacterial suspension was inoculated explant is performed as described in Example 2 embodiment. 细菌终浓度调整为OD660=0.55或0.85。 Bacterial concentration was adjusted to a final OD660 = 0.55 or 0.85.

接种步骤之后,将外植体转移到含有20g/L蔗糖或15g/L甘露糖的共培养培养基中,并保持在20-23℃16小时光/8小时黑暗条件下培养。 After the inoculation step, the explants were transferred to a solution containing 20g / L sucrose or 15g / L mannose co-culture medium and maintained in culture at 20-23 ℃ 16 hour light / 8 h dark conditions.

共培养3-5天后,使用荧光显微镜观察荧光蛋白基因的表达。 After 3-5 days co-cultivation, using a fluorescent microscope Fluorescent Protein Gene Expression. 在含有甘露糖的共培养培养基中接种农杆菌的外植体,与含有蔗糖的共培养培养基中共培养的农杆菌接种的外植体相比,显示至少两倍的荧光斑点数。 The mannose-containing medium explants inoculated with Agrobacterium co-cultivation, compared to the co-cultivation medium containing sucrose co-cultured explants inoculated with Agrobacterium, the number of fluorescence spots displayed at least twice. 之后的芽再生和选择步骤如实施例2中所述方法进行。 After selection and shoot regeneration step as described in Example 2 of the method.

在共培养培养基含有甘露糖的实验中发现转化芽的产量显著地增加了(表3)。 It found that the conversion of shoots in co-cultivation medium containing mannose in experimental production significantly increased (Table 3). 从含有甘露糖的共培养培养基中有五个转化的芽生根,并转移到了土壤中。 Mannose-containing co-cultivation medium with a five transformed shoots rooted and transferred to soil from. 之后Taqman和Southern印迹分析证实有转基因的整合。 After Taqman and Southern blot analysis confirmed the integration of the transgene. 在T1子代中转基因的表达证实了这些转移基因的种系传递。 It confirmed the expression of transgenes germline transmission of these genes in the T1 progeny transfer.

表3 表达ZsGreen1荧光蛋白基因的转化芽,其中外植体和农杆菌在甘露糖或蔗糖中共培养 Conversion Table shoots ZsGreen1 Fluorescent Protein Gene Expression 3, wherein the explant and Agrobacterium cultures mannose or sucrose CCP

实施例5在本实施例中,使用含有质粒pNOV2105(SMAS-PMI SMAS-GUS,如实施例1中所述)的农杆菌EHA101用于大豆转化。 Agrobacterium Example 5 In this example, a plasmid containing pNOV2105 (SMAS-PMI SMAS-GUS, as described in Example 1) of EHA101 for soybean transformation. 外植体的制备、农杆菌悬液以及农杆菌接种外植体与实施例2中所述的相同。 Preparation of explants inoculated with Agrobacterium Agrobacterium suspension and explant in Example 2 above. 细菌终浓度调整为OD660=0.45或0.6。 Bacterial concentration was adjusted to a final OD660 = 0.45 or 0.6.

农杆菌接种后,将外植体转移到含有20g/L蔗糖或15g/L甘露糖的共培养培养基中。 After Agrobacterium inoculation, the explants were transferred to a solution containing 20g / L sucrose or 15g / L co-cultivation medium mannose. 在20-23℃16小时光/8小时黑暗条件下进行共培养。 Co-cultured at 20-23 ℃ 16 hour light / 8 h dark conditions. 共培养3-5天后,用组织化学gus测定方法观察GUS基因的表达。 After 3-5 days co-culture, the expression of the GUS gene was observed by histochemical GUS assay method. 在含有甘露糖的共培养培养基中共培养的外植体,与在含有蔗糖的共培养培养基中共培养的外植体相比,显示至少两倍的GUS斑点数。 Mannose-containing culture medium were co-cultured explants compared to the co-cultivation medium containing sucrose co-cultured explants showing GUS at least twice the number of spots. 芽再生和选择如实施例2中所述方法进行。 Shoot regeneration and selection as described in Example 2 carried out. 在共培养培养基中加入甘露糖的实验中观察到转化芽的产量显著增加(表4)。 Mannose is added in co-culture experiments, medium conversion was observed a significant increase in shoot yield (Table 4).

表4 表达GUS基因的转化芽 Table 4 Conversion shoots GUS gene expression

实施例6 Example 6

在本实施例中,使用含有质粒pBSC11234(图5)的农杆菌EHA101用于大豆转化。 In the present embodiment, a plasmid containing pBSC11234 (FIG. 5) of Agrobacterium EHA101 for soybean transformation. pBSC11234的组成和序列如SEQ ID NO:3所示。 PBSC11234 composition and sequence as SEQ ID NO: 3 shown in FIG. pBSC11234含有CMP-PMI:β-伴大豆球蛋白(conglycinin)-半乳糖苷酶基因构建体。 pBSC11234 containing CMP-PMI: β- conglycinin (conglycinin) - galactosidase gene construct. 外植体的制备、农杆菌悬液以及农杆菌接种外植体与实施例2中所述的相同。 Preparation of explants inoculated with Agrobacterium Agrobacterium suspension and explant in Example 2 above. 细菌终浓度调整为OD660=0.6。 Bacterial concentration was adjusted to a final OD660 = 0.6. 该共培养液体培养基含有B5盐(0.1×)、B5维生素(1×)、乙酰丁香酮80mg/L、蔗糖20g/L、BAP 2mg/L、GA30.25mg/L、MES 3.9g/L以及pH5.4。 The liquid co-cultivation medium containing B5 salts (0.1 ×), B5 vitamins (1 ×), acetosyringone 80mg / L, sucrose 20g / L, BAP 2mg / L, GA30.25mg / L, MES 3.9g / L and pH5.4. 通过在液体共培养培养基中掺入5g/L精制琼脂制备固体共培养培养基。 Medium by incorporating 5g / L purified agar preparing solid co-cultivation medium in liquid co-cultivation.

农杆菌接种后,将外植体转移到固体共培养培养基上,在20-24℃16小时光/8小时黑暗条件下培养。 After Agrobacterium inoculation, the explants are transferred to a solid co-cultivation medium and incubated at 20-24 ℃ 16 hour light / 8 h dark conditions. 共培养3-5天后,去掉初生和次生芽分生组织并丢弃,将所得到的外植体转移到REG-4培养基中。 After 3-5 days co-cultivation, remove the primary and secondary shoot meristem and discarded, the transfer of the explants obtained REG-4 to the medium. REG-4培养基含有B5盐(1×)、B5维生素(1×)、BAP 1mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、氨噻肟头孢霉素100mg/L、替卡西林300mg/L、甘露糖15-20g/L、蔗糖0、0.25或1g/L、精制琼脂10g/L以及pH5.6。5-7天期间之后,去除从接近子叶的腋生分生组织长出的任何芽,然后将外植体转移到REG-5培养基中,REG-5培养基含有B5盐(1×)、B5维生素(1×)、BAP 0.5mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、氨噻肟头孢霉素100mg/L、替卡西林300mg/L、甘露糖15-20g/L、蔗糖1g/L、精制琼脂10g/L以及pH5.6。 REG-4 medium containing B5 salts (1 ×), B5 vitamins (1 ×), BAP 1mg / L, glutamine, 50mg / L, asparagine 50mg / L, cefotaxime cefotaxime 100mg / L, for ticarcillin 300mg / L, mannose 15-20g / L, then /, purified agar 10g / L sucrose 0, 0.25, or during and 1g L pH5.6.5-7 days, removed from the proximity of the axillary meristematic tissue ingrowth cotyledon any budding, and the explants are transferred to medium REG-5, REG-5 medium containing B5 salts (1 ×), B5 vitamins (1 ×), BAP 0.5mg / L, glutamine 50mg / L, asparagine 50mg / L, cefotaxime cefotaxime 100mg / L, ticarcillin 300mg / L, mannose 15-20g / L, sucrose 1g / L, purified agar 10g / L and pH5.6. 在四周时,将外植体转移到REG-6培养基中以便芽伸长。 In four weeks, the explants are transferred to the REG-6 medium for shoot elongation. REG-6培养基含有MS盐(1×)、MS维生素(1×)(MS维生素组合物:肌醇100mg/L、烟酸0.5mg/L、盐酸吡哆醇0.5mg/L、盐酸硫胺素0.1mg/L、甘氨酸2mg/L)、肌醇200mg/L、BAP 0.2mg/L、玉米素核糖核苷0.5mg/L、IBA 0.1mg/L、GA31mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、替卡西林300mg/L、甘露糖15g/L、蔗糖5g/L、硝酸银0.8mg/L、精制琼脂10g/L、以及pH5.6。 REG-6 medium containing MS salts (1 ×), MS vitamins (1 ×) (MS vitamins composition: myo-inositol 100mg / L, nicotinic acid 0.5mg / L, pyridoxine hydrochloride 0.5mg / L, thiamine hydrochloride Su 0.1mg / L, glycine 2mg / L), myo-inositol 200mg / L, BAP 0.2mg / L, zeatin riboside 0.5mg / L, IBA 0.1mg / L, GA31mg / L, glutamine, 50mg / L , asparagine, 50mg / L, ticarcillin 300mg / L, mannose 15g / L, sucrose 5g / L, silver nitrate 0.8mg / L, purified agar 10g / L, and pH5.6. 每两周将外植体转移到新鲜的REG-6培养基中。 Every two weeks explants were transferred to fresh medium REG-6. 将伸长的芽(2-4cm长)移出,并在生根培养基中生根,然后转移到土壤中。 The elongated shoots (2-4cm long) was removed, and rooting rooting medium, then transferred to soil. 生根培养基含有MS盐(1×)、B5维生素(1×)、谷氨酰胺100mg/L、天冬酰胺100mg/L、IBA 0.7mg/L、timentin 100mg/L、以及蔗糖15g/L。 Rooting medium containing MS salts (1 ×), B5 vitamins (1 ×), Glutamine 100mg / L, asparagine 100mg / L, IBA 0.7mg / L, timentin 100mg / L, sucrose and 15g / L. Taqman分析证实在两个事件的叶子样本中有转基因(α半乳糖苷酶和磷酸甘露糖异构酶)的存在。 Taqman analysis confirmed the presence of the transgene ([alpha] -galactosidase and phosphate isomerase) in the two leaf sample event.

实施例7在本实施例中,使用含有质粒pBSC11369(图6)的农杆菌EHA101用于大豆转化。 Example 7 In this example, a plasmid containing Agrobacterium EHA101 pBSC11369 (FIG. 6) is used for soybean transformation. pBSC11369的组成和序列如SEQ ID NO:4所示。 PBSC11369 composition and sequence as SEQ ID NO: 4 shown in FIG. pBSC11369含有CMP-HPT:CMP-ZsGreen1基因构建体。 pBSC11369 containing CMP-HPT: CMP-ZsGreen1 gene construct. 外植体的制备、农杆菌悬液以及农杆菌接种外植体与实施例2中所述的相同。 Preparation of explants inoculated with Agrobacterium Agrobacterium suspension and explant in Example 2 above. 细菌终浓度调整为OD660=0.6。 Bacterial concentration was adjusted to a final OD660 = 0.6. 所述共培养液体培养基含有B5盐(0.1×)、B5维生素(1×)、乙酰丁香酮80mg/L、蔗糖20g/L、BAP 2mg/L、GA30.25mg/L、MES 3.9g/L以及pH5.4。 The liquid co-culture medium containing B5 salts (0.1 ×), B5 vitamins (1 ×), acetosyringone 80mg / L, sucrose 20g / L, BAP 2mg / L, GA30.25mg / L, MES 3.9g / L and pH5.4. 通过在液体共培养培养基中掺入5g/L精制琼脂制备固体共培养培养基。 Medium by incorporating 5g / L purified agar preparing solid co-cultivation medium in liquid co-cultivation.

农杆菌接种后,将外植体转移到固体共培养培养基上,在20-24℃16小时光/8小时黑暗条件下培养。 After Agrobacterium inoculation, the explants are transferred to a solid co-cultivation medium and incubated at 20-24 ℃ 16 hour light / 8 h dark conditions. 共培养3-5天后,为促进芽从初生叶基部区生长,从外植体上去除初生和次生分生组织,之后将外植体转移到REG-7培养基中。 After 3-5 days co-culture, to promote shoot growth from the primary leaf base region, removing the primary and secondary meristem explants from, after which the explants are transferred to the REG-7 medium. REG-7培养基含有B5盐(1×)、B5维生素(1×)、BAP 1mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、氨噻肟头孢霉素100mg/L、替卡西林300mg/L、蔗糖30g/L、潮霉素2-5mg/L、精制琼脂10g/L以及pH5.6。 REG-7 medium containing B5 salts (1 ×), B5 vitamins (1 ×), BAP 1mg / L, glutamine, 50mg / L, asparagine 50mg / L, cefotaxime cefotaxime 100mg / L, for ticarcillin 300mg / L, sucrose 30g / L, hygromycin 2-5mg / L, purified agar 10g / L and pH5.6. 将外植体直立放置以便将外植体的上胚轴端插入培养基中。 The explants were placed upright to the upper end of the hypocotyl explants inserted medium. 7-10天期间之后,去除从靠近子叶的腋生分生组织长出的所有芽。 During and after 7-10 days, removed from all the axillary buds near the cotyledon meristem grow. 将外植体转移到新鲜REG-8培养基中,REG-8培养基含有B5盐(1×)、B5维生素(1×)、BAP 0.5mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、氨噻肟头孢霉素100mg/L、替卡西林300mg/L、蔗糖30g/L、精制琼脂10g/L以及pH在5.6。 The explants were transferred to fresh medium REG-8, REG-8 medium containing B5 salts (1 ×), B5 vitamins (1 ×), BAP 0.5mg / L, glutamine, 50mg / L, asparagine 50mg / L, cefotaxime cefotaxime 100mg / L, ticarcillin 300mg / L, sucrose 30g / L, purified agar 10g / L and a pH of 5.6. 再两周之后,将外植体转移到REG-9培养基中,然后每两周传代培养。 After another two weeks, the explants are transferred to the REG-9 culture medium, and subcultured every two weeks. REG-9培养基含有MS盐(1×)、MS维生素(1×)、肌醇200mg/L、BAP 0.2mg/L、玉米素核糖核苷0.5mg/L、IBA 0.1mg/L、GA31mg/L、谷氨酰胺50mg/L、天冬酰胺50mg/L、硝酸银0.8mg/L、替卡西林300mg/L、蔗糖30g/L、潮霉素0.1-0.2mg/L、精制琼脂10g/L以及pH5.6。 REG-9 medium containing MS salts (1 ×), MS vitamins (1 ×), myo-inositol 200mg / L, BAP 0.2mg / L, zeatin riboside 0.5mg / L, IBA 0.1mg / L, GA31mg / L, glutamine, 50mg / L, asparagine 50mg / L, silver nitrate 0.8mg / L, ticarcillin 300mg / L, sucrose 30g / L, hygromycin 0.1-0.2mg / L, purified agar 10g / L and pH5.6. 将伸长的芽(2-4cm长)移出,在生根培养基中生根,然后转移到土壤中。 The elongated shoots (2-4cm long) are removed, rooting rooting medium, then transferred to soil. 生根培养基含有MS盐(1×)、B5维生素(1×)、谷氨酰胺100mg/L、天冬酰胺100mg/L、IBA 0.7mg/L、timentin 100mg/L、以及蔗糖15g/L。 Rooting medium containing MS salts (1 ×), B5 vitamins (1 ×), Glutamine 100mg / L, asparagine 100mg / L, IBA 0.7mg / L, timentin 100mg / L, sucrose and 15g / L. Taqman分析证实从五个事件获得的叶子样本中有转移基因(HPT以及ZsGreen1)的存在。 Taqman analysis confirmed from leaf samples obtained from five events in the presence of metastasis gene (HPT and ZsGreen1) of. 通过在荧光显微镜下观察证实了在植物部分中有ZsGreen1基因的表达。 By observation under a fluorescent microscope demonstrated ZsGreen1 gene expression in plant parts.

本文引用的所有出版物、专利及专利申请作为参考文献并入本文。 All publications cited herein, patents and patent applications are incorporated herein by reference. 尽管在前面的说明书中,描述了本发明有关的某些优选实施方案以及为了举例说明陈述了许多细节,但对于本领域的技术人员显而易见的是本发明可以有另外的实施方案以及本文所描述的某些细节可以有相当大的变化而不偏离本发明的基本原则。 While in the foregoing specification, are described certain preferred embodiments of the present invention is related to the purpose of illustration and many details are set forth, but for those skilled in the art will be apparent that the present invention may be other embodiments and the embodiment described herein certain details may have considerable variation without departing from the basic principles of the invention.

序列表<110>Syngenta Participations AG<120>转化大豆的方法<130>70094 USPS<160>4<170>PatentIn version 3.2<210>1<211>9555<212>DNA<213>人工<220> SEQUENCE LISTING & lt; 110 & gt; Syngenta Participations AG & lt; 120 & gt; The method of transformation of soybean & lt; 130 & gt; 70094 USPS & lt; 160 & gt; 4 & lt; 170 & gt; PatentIn version 3.2 & lt; 210 & gt; 1 & lt; 211 & gt; 9555 & lt; 212 & gt; DNA & lt; 213 & gt; artificial & lt ; 220 & gt;

<223>pNOV2145<400>1gatccaccgg tcgccaccat ggcccagtcc aagcacggcc tgaccaagga gatgaccatg 60aagtaccgca tggagggctg cgtggacggc cacaagttcg tgatcaccgg cgagggcatc 120ggctacccct tcaagggcaa gcaggccatc aacctgtgcg tggtggaggg cggccccttg 180cccttcgccg aggacatctt gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag 240tacccccagg acatcgtcga ctacttcaag aactcctgcc ccgccggcta cacctgggac 300cgctccttcc tgttcgagga cggcgccgtg tgcatctgca acgccgacat caccgtgagc 360gtggaggaga actgcatgta ccacgagtcc aagttctacg gcgtgaactt ccccgccgac 420ggccccgtga tgaagaagat gaccgacaac tgggagccct cctgcgagaa gatcatcccc 480gtgcccaagc agggcatctt gaagggcgac gtgagcatgt acctgctgct gaaggacggt 540ggccgcttgc gctgccagtt cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg 600cccgactggc acttcatcca gcacaagctg acccgcgagg accgcagcga cgccaagaac 660cagaagtggc acctgaccga gcacgccatc gcctccggct ccgccttgcc ctgagcggcc 720ctctagatcc ccgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt 780gaatcctgtt gccggtcttg cgatgattat & Lt; 223 & gt; pNOV2145 & lt; 400 & gt; 1gatccaccgg tcgccaccat ggcccagtcc aagcacggcc tgaccaagga gatgaccatg 60aagtaccgca tggagggctg cgtggacggc cacaagttcg tgatcaccgg cgagggcatc 120ggctacccct tcaagggcaa gcaggccatc aacctgtgcg tggtggaggg cggccccttg 180cccttcgccg aggacatctt gtccgccgcc ttcatgtacg gcaaccgcgt gttcaccgag 240tacccccagg acatcgtcga ctacttcaag aactcctgcc ccgccggcta cacctgggac 300cgctccttcc tgttcgagga cggcgccgtg tgcatctgca acgccgacat caccgtgagc 360gtggaggaga actgcatgta ccacgagtcc aagttctacg gcgtgaactt ccccgccgac 420ggccccgtga tgaagaagat gaccgacaac tgggagccct cctgcgagaa gatcatcccc 480gtgcccaagc agggcatctt gaagggcgac gtgagcatgt acctgctgct gaaggacggt 540ggccgcttgc gctgccagtt cgacaccgtg tacaaggcca agtccgtgcc ccgcaagatg 600cccgactggc acttcatcca gcacaagctg acccgcgagg accgcagcga cgccaagaac 660cagaagtggc acctgaccga gcacgccatc gcctccggct ccgccttgcc ctgagcggcc 720ctctagatcc ccgaatttcc ccgatcgttc aaacatttgg caataaagtt tcttaagatt 780gaatcctgtt gccggtcttg cgatgattat catataattt ctgttgaatt acgttaagca 840tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt 900cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 960attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttgggtaccg aattcactgg 1020ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 1080cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 1140cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 1200atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg 1260 catataattt ctgttgaatt acgttaagca 840tgtaataatt aacatgtaat gcatgacgtt atttatgaga tgggttttta tgattagagt 900cccgcaatta tacatttaat acgcgataga aaacaaaata tagcgcgcaa actaggataa 960attatcgcgc gcggtgtcat ctatgttact agatcgggaa ttgggtaccg aattcactgg 1020ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg 1080cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt 1140cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc 1200atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg 1260

catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 1320tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 1380ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 1440tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 1500atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 1560tgagacaata accctgataa atgcttcaat ggcgcgccgg taccagcttg catgcctgca 1620ggtcgactct agaggatcct ggcagacaaa gtggcagaca tactgtccca caaatgaaga 1680tggaatctgt aaaagaaaac gcgtgaaata atgcgtctga caaaggttag gtcggctgcc 1740tttaatcaat accaaagtgg tccctaccac gatggaaaaa ctgtgcagtc ggtttggctt 1800tttctgacga acaaataaga ttcgtggccg acaggtgggg gtccaccatg tgaaggcatc 1860ttcagactcc aataatggag caatgacgta agggcttacg aaataagtaa gggtagtttg 1920ggaaatgtcc actcacccgt cagtctataa atacttagcc cctccctcat tgttaaggga 1980gcaaaatctc agagagatag tcctagagag agaaagagag caagtagcct agaagtagga 2040tccccgatca tgcaaaaact cattaactca gtgcaaaact atgcctg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc 1320tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga 1380ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt 1440tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa 1500atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca 1560tgagacaata accctgataa atgcttcaat ggcgcgccgg taccagcttg catgcctgca 1620ggtcgactct agaggatcct ggcagacaaa gtggcagaca tactgtccca caaatgaaga 1680tggaatctgt aaaagaaaac gcgtgaaata atgcgtctga caaaggttag gtcggctgcc 1740tttaatcaat accaaagtgg tccctaccac gatggaaaaa ctgtgcagtc ggtttggctt 1800tttctgacga acaaataaga ttcgtggccg acaggtgggg gtccaccatg tgaaggcatc 1860ttcagactcc aataatggag caatgacgta agggcttacg aaataagtaa gggtagtttg 1920ggaaatgtcc actcacccgt cagtctataa atacttagcc cctccctcat tgttaaggga 1980gcaaaatctc agagagatag tcctagagag agaaagagag caagtagcct agaagtagga 2040tccccgatca tgcaaaaact cattaactca gtgcaaaact atgcctg ggg cagcaaaacg 2100gcgttgactg aactttatgg tatggaaaat ccgtccagcc agccgatggc cgagctgtgg 2160atgggcgcac atccgaaaag cagttcacga gtgcagaatg ccgccggaga tatcgtttca 2220ctgcgtgatg tgattgagag tgataaatcg actctgctcg gagaggccgt tgccaaacgc 2280tttggcgaac tgcctttcct gttcaaagta ttatgcgcag cacagccact ctccattcag 2340gttcatccaa acaaacacaa ttctgaaatc ggttttgcca aagaaaatgc cgcaggtatc 2400ccgatggatg ccgccgagcg taactataaa gatcctaacc acaagccgga gctggttttt 2460gcgctgacgc ctttccttgc gatgaacgcg tttcgtgaat tttccgagat tgtctcccta 2520ctccagccgg tcgcaggtgc acatccggcg attgctcact ttttacaaca gcctgatgcc 2580gaacgtttaa gcgaactgtt cgccagcctg ttgaatatgc agggtgaaga aaaatcccgc 2640gcgctggcga ttttaaaatc ggccctcgat agccagcagg gtgaaccgtg gcaaacgatt 2700cgtttaattt ctgaatttta cccggaagac agcggtctgt tctccccgct attgctgaat 2760gtggtgaaat tgaaccctgg cgaagcgatg ttcctgttcg ctgaaacacc gcacgcttac 2820ctgcaaggcg tggcgctgga agtgatggca aactccgata acgtgctgcg tgcgggtctg 2880acgcctaaat acattgatat tccggaa ggg cagcaaaacg 2100gcgttgactg aactttatgg tatggaaaat ccgtccagcc agccgatggc cgagctgtgg 2160atgggcgcac atccgaaaag cagttcacga gtgcagaatg ccgccggaga tatcgtttca 2220ctgcgtgatg tgattgagag tgataaatcg actctgctcg gagaggccgt tgccaaacgc 2280tttggcgaac tgcctttcct gttcaaagta ttatgcgcag cacagccact ctccattcag 2340gttcatccaa acaaacacaa ttctgaaatc ggttttgcca aagaaaatgc cgcaggtatc 2400ccgatggatg ccgccgagcg taactataaa gatcctaacc acaagccgga gctggttttt 2460gcgctgacgc ctttccttgc gatgaacgcg tttcgtgaat tttccgagat tgtctcccta 2520ctccagccgg tcgcaggtgc acatccggcg attgctcact ttttacaaca gcctgatgcc 2580gaacgtttaa gcgaactgtt cgccagcctg ttgaatatgc agggtgaaga aaaatcccgc 2640gcgctggcga ttttaaaatc ggccctcgat agccagcagg gtgaaccgtg gcaaacgatt 2700cgtttaattt ctgaatttta cccggaagac agcggtctgt tctccccgct attgctgaat 2760gtggtgaaat tgaaccctgg cgaagcgatg ttcctgttcg ctgaaacacc gcacgcttac 2820ctgcaaggcg tggcgctgga agtgatggca aactccgata acgtgctgcg tgcgggtctg 2880acgcctaaat acattgatat tccggaa ctg gttgccaatg tgaaattcga agccaaaccg 2940gctaaccagt tgttgaccca gccggtgaaa caaggtgcag aactggactt cccgattcca 3000gtggatgatt ttgccttctc gctgcatgac cttagtgata aagaaaccac cattagccag 3060cagagtgccg ccattttgtt ctgcgtcgaa ggcgatgcaa cgttgtggaa aggttctcag 3120cagttacagc ttaaaccggg tgaatcagcg tttattgccg ccaacgaatc accggtgact 3180 ctg gttgccaatg tgaaattcga agccaaaccg 2940gctaaccagt tgttgaccca gccggtgaaa caaggtgcag aactggactt cccgattcca 3000gtggatgatt ttgccttctc gctgcatgac cttagtgata aagaaaccac cattagccag 3060cagagtgccg ccattttgtt ctgcgtcgaa ggcgatgcaa cgttgtggaa aggttctcag 3120cagttacagc ttaaaccggg tgaatcagcg tttattgccg ccaacgaatc accggtgact 3180

gtcaaaggcc acggccgttt agcgcgtgtt tacaacaagc tgtaagagct tactgaaaaa 3240attaacatct cttgctaagc tgggagctct agatccccga atttccccga tcgttcaaac 3300atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 3360taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 3420atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 3480aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 3540cgggaattgg gtaccatgcc cgggcggcca gcatggccgt atccgcaatg tgttattaag 3600ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca gccaacagct 3660ccccgaccgg cagctcggca caaaatcacc actcgataca ggcagcccat cagaattaat 3720tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca 3780gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct 3840caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa 3900atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg 3960agcggataac aatttcacac aggaaacaga ccatgaggga agcgttg gtcaaaggcc acggccgttt agcgcgtgtt tgtaagagct tactgaaaaa 3240attaacatct cttgctaagc tgggagctct agatccccga atttccccga tcgttcaaac 3300atttggcaat aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata 3360taatttctgt tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt 3420atgagatggg tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac 3480aaaatatagc gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat 3540cgggaattgg gtaccatgcc cgggcggcca gcatggccgt atccgcaatg tgttattaag 3600ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca gccaacagct 3660ccccgaccgg cagctcggca tacaacaagc caaaatcacc actcgataca ggcagcccat cagaattaat 3720tctcatgttt gacagcttat catcgactgc acggtgcacc aatgcttctg gcgtcaggca 3780gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc actgcataat tcgtgtcgct 3840caaggcgcac tcccgttctg gataatgttt tttgcgccga catcataacg gttctggcaa 3900atattctgaa atgagctgtt gacaattaat catccggctc gtataatgtg tggaattgtg 3960agcggataac aatttcacac aggaaacaga ccatgaggga agcgttg atc gccgaagtat 4020cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg 4080ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt 4140tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc 4200ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca 4260ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg 4320gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg 4380atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg 4440cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct 4500taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt 4560tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg 4620actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctaggcagg 4680cttatcttgg acaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgttc 4740actacgtgaa aggcgagatc accaaagtag tcggcaaata aagctctagt ggatctccgt 4800acccccgggg gatctggctc gcggcgg atc gccgaagtat 4020cgactcaact atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg 4080ccgtacattt gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt 4140tgctggttac ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc 4200ttttggaaac ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca 4260ttgttgtgca cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg 4320gagaatggca gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg 4380atctggctat cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg 4440cggaggaact ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct 4500taacgctatg gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt 4560tgtcccgcat ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg 4620actgggcaat ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctaggcagg 4680cttatcttgg acaagaagat cgcttggcct cgcgcgcaga tcagttggaa gaatttgttc 4740actacgtgaa aggcgagatc accaaagtag tcggcaaata aagctctagt ggatctccgt 4800acccccgggg gatctggctc gcggcgg acg cacgacgccg gggcgagacc ataggcgatc 4860tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt 4920tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca gccgcaattc 4980tgacgaactg cccatttagc tggagatgat tgtacatcct tcacgtgaaa atttctcaag 5040 acg cacgacgccg gggcgagacc ataggcgatc 4860tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt 4920tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca gccgcaattc 4980tgacgaactg cccatttagc tggagatgat tgtacatcct tcacgtgaaa atttctcaag 5040

cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac acgttcttct 5100tgtcgatgac gacgtcgcta tgcggcatct tattattgaa taccttacga tccacgcctt 5160caaagtgacc gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt 5220cgatgtcgtg gttgttgatc taaatttagg tcgtgaagat gggctcgaga tcgttcgtaa 5280tctggcggca aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac 5340ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc cgttcagtat 5400cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc 5460caaagaccga cggtcttttt gttttactga ctggacactt aatctcaggc aacgtcgctt 5520gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc ttctcctcgc 5580gtttttagag aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt 5640acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc tgcgccgcaa 5700acttgaggca gatccgtcaa gccctcaact gataaaaaca gcaagaggtg ccggttattt 5760ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatg gcagcctgag ccaattccca 5820gatccccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggt cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac acgttcttct 5100tgtcgatgac gacgtcgcta tgcggcatct tattattgaa taccttacga tccacgcctt 5160caaagtgacc gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt 5220cgatgtcgtg gttgttgatc taaatttagg tcgtgaagat gggctcgaga tcgttcgtaa 5280tctggcggca aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac 5340ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc cgttcagtat 5400cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc 5460caaagaccga cggtcttttt gttttactga ctggacactt aatctcaggc aacgtcgctt 5520gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc ttctcctcgc 5580gtttttagag aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt 5640acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc tgcgccgcaa 5700acttgaggca gatccgtcaa gccctcaact gataaaaaca gcaagaggtg ccggttattt 5760ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatg gcagcctgag ccaattccca 5820gatccccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggt aca aatcggcgcg 5880gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc 5940atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa 6000gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac 6060gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc 6120atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc 6180cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt 6240gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga 6300taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta 6360ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc 6420attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc 6480ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa 6540accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca 6600gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc 6660atcggccgtt ttctctaccg cctggca aca aatcggcgcg 5880gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc 5940atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa 6000gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac 6060gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc 6120atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc 6180cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt 6240gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga 6300taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta 6360ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc 6420attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc 6480ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa 6540accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca 6600gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat cgatcccggc 6660atcggccgtt ttctctaccg cctggca cgc cgcgccgcag gcaaggcaga agccagatgg 6720ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc 6780accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg 6840gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc 6900gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt 6960 cgc cgcgccgcag gcaaggcaga agccagatgg 6720ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc 6780accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg 6840gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc 6900gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt 6960

cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg 7020aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa 7080gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt 7140aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag 7200ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg 7260cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg 7320cgcggacaag ccgcgccgtc gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa 7380ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag 7440ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt 7500gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa 7560gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt 7620acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt 7680tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag 7740aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcgg cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg 7020aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa 7080gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt 7140aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag 7200ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg 7260cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg 7320cgcggacaag ccgcgccgtc gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa 7380ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag 7440ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt 7500gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa 7560gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt 7620acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt 7680tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttctgt aatgaaggag 7740aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcgg tct gcgattccga 7800ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg 7860agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctct gcattaatga 7920atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7980actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 8040gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 8100cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 8160ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 8220ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 8280ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 8340agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 8400cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 8460aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 8520gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact 8580agaagaacag tatttggtat ctgcgct tct gcgattccga 7800ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg 7860agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctct gcattaatga 7920atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7980actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 8040gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 8100cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 8160ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 8220ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 8280ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 8340agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 8400cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 8460aacccggtaa ggcctaacta cggctacact 8580agaagaacag gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 8520gcgaggtatg taggcggtgc tacagagttc ttgaagtggt tatttggtat ctgcgct ctg ctgaagccag ttaccttcgg aaaaagagtt 8640ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8700cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8760tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8820 ctg ctgaagccag ttaccttcgg aaaaagagtt 8640ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8700cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8760tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8820

aggatcttca cctagatcct tttgatccgg aattaattcc tgtggttggc atgcacatac 8880aaatggacga acggataaac cttttcacgc ccttttaaat atccgattat tctaataaac 8940gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 9000aaggcgggaa acgacaatct gatcatgagc ggagaattaa gggagtcacg ttatgacccc 9060cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc aacgctgcag 9120gaattggccg cagcggccat ttaaatcaat tgggcgcgta cgtagcacta gtgcgcgatc 9180gcttaattaa gcggcgcgcc taaagcttct ggcagacaaa gtggcagaca tactgtccca 9240caaatgaaga tggaatctgt aaaagaaaac gcgtgaaata atgcgtctga caaaggttag 9300gtcggctgcc tttaatcaat accaaagtgg tccctaccac gatggaaaaa ctgtgcagtc 9360ggtttggctt tttctgacga acaaataaga ttcgtggccg acaggtgggg gtccaccatg 9420tgaaggcatc ttcagactcc aataatggag caatgacgta agggcttacg aaataagtaa 9480gggtagtttg ggaaatgtcc actcacccgt cagtctataa atacttagcc cctccctcat 9540tgttaaggga gcaag 9555<210>2<211>9546<212>DNA<213&gt aggatcttca cctagatcct tttgatccgg aattaattcc tgtggttggc atgcacatac 8880aaatggacga acggataaac cttttcacgc ccttttaaat atccgattat tctaataaac 8940gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 9000aaggcgggaa acgacaatct gatcatgagc ggagaattaa gggagtcacg ttatgacccc 9060cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc aacgctgcag 9120gaattggccg cagcggccat ttaaatcaat tgggcgcgta cgtagcacta gtgcgcgatc 9180gcttaattaa gcggcgcgcc taaagcttct ggcagacaaa gtggcagaca tactgtccca 9240caaatgaaga tggaatctgt aaaagaaaac gcgtgaaata atgcgtctga caaaggttag 9300gtcggctgcc tttaatcaat accaaagtgg tccctaccac gatggaaaaa ctgtgcagtc 9360ggtttggctt tttctgacga acaaataaga ttcgtggccg acaggtgggg gtccaccatg 9420tgaaggcatc ttcagactcc aataatggag caatgacgta agggcttacg aaataagtaa 9480gggtagtttg ggaaatgtcc actcacccgt cagtctataa atacttagcc cctccctcat 9540tgttaaggga gcaag 9555 & lt; 210 & gt; 2 & lt; 211 & gt; 9546 & lt; 212 & gt; DNA & lt; 213 & gt ;人工<220> ; Artificial & lt; 220 & gt;

<223>pNOV2147<400>2ggatccccga tcatgcaaaa actcattaac tcagtgcaaa actatgcctg gggcagcaaa 60acggcgttga ctgaacttta tggtatggaa aatccgtcca gccagccgat ggccgagctg 120tggatgggcg cacatccgaa aagcagttca cgagtgcaga atgccgccgg agatatcgtt 180tcactgcgtg atgtgattga gagtgataaa tcgactctgc tcggagaggc cgttgccaaa 240cgctttggcg aactgccttt cctgttcaaa gtattatgcg cagcacagcc actctccatt 300caggttcatc caaacaaaca caattctgaa atcggttttg ccaaagaaaa tgccgcaggt 360atcccgatgg atgccgccga gcgtaactat aaagatccta accacaagcc ggagctggtt 420tttgcgctga cgcctttcct tgcgatgaac gcgtttcgtg aattttccga gattgtctcc 480ctactccagc cggtcgcagg tgcacatccg gcgattgctc actttttaca acagcctgat 540gccgaacgtt taagcgaact gttcgccagc ctgttgaata tgcagggtga agaaaaatcc 600cgcgcgctgg cgattttaaa atcggccctc gatagccagc agggtgaacc gtggcaaacg 660attcgtttaa tttctgaatt ttacccggaa gacagcggtc tgttctcccc gctattgctg 720aatgtggtga aattgaaccc tggcgaagcg atgttcctgt tcgctgaaac accgcacgct 780tacctgcaag gcgtggcgct ggaagtgatg & Lt; 223 & gt; pNOV2147 & lt; 400 & gt; 2ggatccccga tcatgcaaaa actcattaac tcagtgcaaa actatgcctg gggcagcaaa 60acggcgttga ctgaacttta tggtatggaa aatccgtcca gccagccgat ggccgagctg 120tggatgggcg cacatccgaa aagcagttca cgagtgcaga atgccgccgg agatatcgtt 180tcactgcgtg atgtgattga gagtgataaa tcgactctgc tcggagaggc cgttgccaaa 240cgctttggcg aactgccttt cctgttcaaa gtattatgcg cagcacagcc actctccatt 300caggttcatc caaacaaaca caattctgaa atcggttttg ccaaagaaaa tgccgcaggt 360atcccgatgg atgccgccga gcgtaactat aaagatccta accacaagcc ggagctggtt 420tttgcgctga cgcctttcct tgcgatgaac 480ctactccagc gcgtttcgtg aattttccga gattgtctcc tcgctgaaac accgcacgct 780tacctgcaag cggtcgcagg tgcacatccg gcgattgctc actttttaca acagcctgat 540gccgaacgtt taagcgaact gttcgccagc ctgttgaata tgcagggtga agaaaaatcc 600cgcgcgctgg cgattttaaa atcggccctc gatagccagc agggtgaacc gtggcaaacg 660attcgtttaa tttctgaatt ttacccggaa gacagcggtc tgttctcccc gctattgctg 720aatgtggtga aattgaaccc tggcgaagcg atgttcctgt gcgtggcgct ggaagtgatg gcaaactccg ataacgtgct gcgtgcgggt 840 gcaaactccg ataacgtgct gcgtgcgggt 840

ctgacgccta aatacattga tattccggaa ctggttgcca atgtgaaatt cgaagccaaa 900ccggctaacc agttgttgac ccagccggtg aaacaaggtg cagaactgga cttcccgatt 960ccagtggatg attttgcctt ctcgctgcat gaccttagtg ataaagaaac caccattagc 1020cagcagagtg ccgccatttt gttctgcgtc gaaggcgatg caacgttgtg gaaaggttct 1080cagcagttac agcttaaacc gggtgaatca gcgtttattg ccgccaacga atcaccggtg 1140actgtcaaag gccacggccg tttagcgcgt gtttacaaca agctgtaaga gcttactgaa 1200aaaattaaca tctcttgcta agctgggagc tctagatccc cgaatttccc cgatcgttca 1260aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 1320atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1380tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1440aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 1500gatcgggaat tgggtaccat gcccgggcgg ccagcatggc cgtatccgca atgtgttatt 1560aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 1620gctccccgac cggcagctcg gcacaaaatc accactcgat acaggca ctgacgccta aatacattga tattccggaa ctggttgcca atgtgaaatt cgaagccaaa 900ccggctaacc agttgttgac ccagccggtg aaacaaggtg cagaactgga cttcccgatt 960ccagtggatg attttgcctt ctcgctgcat gaccttagtg ataaagaaac caccattagc 1020cagcagagtg ccgccatttt gttctgcgtc gaaggcgatg caacgttgtg gaaaggttct 1080cagcagttac agcttaaacc gggtgaatca gcgtttattg ccgccaacga atcaccggtg 1140actgtcaaag gccacggccg tttagcgcgt gtttacaaca agctgtaaga gcttactgaa 1200aaaattaaca tctcttgcta agctgggagc tctagatccc cgaatttccc cgatcgttca 1260aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc gatgattatc 1320atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg catgacgtta 1380tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata cgcgatagaa 1440aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta 1500gatcgggaat tgggtaccat gcccgggcgg ccagcatggc cgtatccgca atgtgttatt 1560aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 1620gctccccgac cggcagctcg gcacaaaatc accactcgat acaggca gcc catcagaatt 1680aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag 1740gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc 1800gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg 1860caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt 1920gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag 1980tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 2040tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 2100atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 2160accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 2220ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 2280ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 2340ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 2400cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 2460ccttaacgct atggaactcg ccgcccg gcc catcagaatt 1680aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag 1740gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc 1800gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg 1860caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtataat gtgtggaatt 1920gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag 1980tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 2040tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 2100atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 2160accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 2220ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 2280ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 2340ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 2400cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 2460ccttaacgct atggaactcg ccgcccg act gggctggcga tgagcgaaat gtagtgctta 2520cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 2580ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 2640aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 2700 act gggctggcga tgagcgaaat gtagtgctta 2520cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 2580ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 2640aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 2700

ttcactacgt gaaaggcgag atcaccaaag tagtcggcaa ataaagctct agtggatctc 2760cgtacccccg ggggatctgg ctcgcggcgg acgcacgacg ccggggcgag accataggcg 2820atctcctaaa tcaatagtag ctgtaacctc gaagcgtttc acttgtaaca acgattgaga 2880atttttgtca taaaattgaa atacttggtt cgcatttttg tcatccgcgg tcagccgcaa 2940ttctgacgaa ctgcccattt agctggagat gattgtacat ccttcacgtg aaaatttctc 3000aagcgctgtg aacaagggtt cagattttag attgaaaggt gagccgttga aacacgttct 3060tcttgtcgat gacgacgtcg ctatgcggca tcttattatt gaatacctta cgatccacgc 3120cttcaaagtg accgcggtag ccgacagcac ccagttcaca agagtactct cttccgcgac 3180ggtcgatgtc gtggttgttg atctaaattt aggtcgtgaa gatgggctcg agatcgttcg 3240taatctggcg gcaaagtctg atattccaat cataattatc agtggcgacc gccttgagga 3300gacggataaa gttgttgcac tcgagctagg agcaagtgat tttatcgcta agccgttcag 3360tatcagagag tttctagcac gcattcgggt tgccttgcgc gtgcgcccca acgttgtccg 3420ctccaaagac cgacggtctt tttgttttac tgactggaca cttaatctca ggcaacgtcg 3480cttgatgtcc gaagctggcg gtgaggtgaa acttacggca ggtgagt ttcactacgt gaaaggcgag atcaccaaag tagtcggcaa ataaagctct agtggatctc 2760cgtacccccg ggggatctgg ctcgcggcgg acgcacgacg ccggggcgag accataggcg 2820atctcctaaa tcaatagtag ctgtaacctc gaagcgtttc acttgtaaca acgattgaga 2880atttttgtca taaaattgaa atacttggtt cgcatttttg tcatccgcgg tcagccgcaa 2940ttctgacgaa ctgcccattt agctggagat gattgtacat ccttcacgtg aaaatttctc 3000aagcgctgtg aacaagggtt cagattttag attgaaaggt gagccgttga aacacgttct 3060tcttgtcgat gacgacgtcg ctatgcggca tcttattatt gaatacctta cgatccacgc 3120cttcaaagtg accgcggtag ccgacagcac ccagttcaca agagtactct cttccgcgac 3180ggtcgatgtc gtggttgttg atctaaattt aggtcgtgaa gatgggctcg agatcgttcg 3240taatctggcg gcaaagtctg atattccaat cataattatc agtggcgacc gccttgagga 3300gacggataaa gttgttgcac tcgagctagg agcaagtgat tttatcgcta agccgttcag 3360tatcagagag tttctagcac gcattcgggt tgccttgcgc gtgcgcccca acgttgtccg 3420ctccaaagac cgacggtctt tttgttttac tgactggaca cttaatctca ggcaacgtcg 3480cttgatgtcc gaagctggcg gtgaggtgaa acttacggca ggtgagt tca atcttctcct 3540cgcgttttta gagaaacccc gcgacgttct atcgcgcgag caacttctca ttgccagtcg 3600agtacgcgac gaggaggttt atgacaggag tatagatgtt ctcattttga ggctgcgccg 3660caaacttgag gcagatccgt caagccctca actgataaaa acagcaagag gtgccggtta 3720tttctttgac gcggacgtgc aggtttcgca cggggggacg atggcagcct gagccaattc 3780ccagatcccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc 3840gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa 3900cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc 3960aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc 4020gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc 4080agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg 4140atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc 4200agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac 4260cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac 4320gtactcaagt tctgccggcg agccgat tca atcttctcct 3540cgcgttttta gagaaacccc gcgacgttct atcgcgcgag caacttctca ttgccagtcg 3600agtacgcgac gaggaggttt atgacaggag tatagatgtt ctcattttga ggctgcgccg 3660caaacttgag gcagatccgt caagccctca actgataaaa acagcaagag gtgccggtta 3720tttctttgac gcggacgtgc aggtttcgca cggggggacg atggcagcct gagccaattc 3780ccagatcccc gaggaatcgg cgtgagcggt cgcaaaccat ccggcccggt acaaatcggc 3840gcggcgctgg gtgatgacct ggtggagaag ttgaaggccg cgcaggccgc ccagcggcaa 3900cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaag cggccgctga tcgaatccgc 3960aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcga ttaggaagcc gcccaagggc 4020gacgagcaac cagatttttt cgttccgatg ctctatgacg tgggcacccg cgatagtcgc 4080agcatcatgg acgtggccgt tttccgtctg tcgaagcgtg accgacgagc tggcgaggtg 4140atccgctacg agcttccaga cgggcacgta gaggtttccg cagggccggc cggcatggcc 4200agtgtgtggg attacgacct ggtactgatg gcggtttccc atctaaccga atccatgaac 4260cgataccggg aagggaaggg agacaagccc ggccgcgtgt tccgtccaca cgttgcggac 4320gtactcaagt tctgccggcg agccgat ggc ggaaagcaga aagacgacct ggtagaaacc 4380tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc 4440cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc 4500gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc 4560acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc 4620 ggc ggaaagcaga aagacgacct ggtagaaacc 4380tgcattcggt taaacaccac gcacgttgcc atgcagcgta cgaagaaggc caagaacggc 4440cgcctggtga cggtatccga gggtgaagcc ttgattagcc gctacaagat cgtaaagagc 4500gaaaccgggc ggccggagta catcgagatc gagctagctg attggatgta ccgcgagatc 4560acagaaggca agaacccgga cgtgctgacg gttcaccccg attacttttt gatcgatccc 4620

ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga 4680tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt 4740ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag 4800gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca 4860tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa 4920ggtcgaaaag gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt 4980gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg 5040taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt 5100attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa 5160gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt 5220cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca 5280gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gctgaggtct gcctcgtgaa 5340gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 5400gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgat ggcatcggcc gttttctcta ccgcctggca cgccgcgccg caggcaaggc agaagccaga 4680tggttgttca agacgatcta cgaacgcagt ggcagcgccg gagagttcaa gaagttctgt 4740ttcaccgtgc gcaagctgat cgggtcaaat gacctgccgg agtacgattt gaaggaggag 4800gcggggcagg ctggcccgat cctagtcatg cgctaccgca acctgatcga gggcgaagca 4860tccgccggtt cctaatgtac ggagcagatg ctagggcaaa ttgccctagc aggggaaaaa 4920ggtcgaaaag gtctctttcc tgtggatagc acgtacattg ggaacccaaa gccgtacatt 4980gggaaccgga acccgtacat tgggaaccca aagccgtaca ttgggaaccg gtcacacatg 5040taagtgactg atataaaaga gaaaaaaggc gatttttccg cctaaaactc tttaaaactt 5100attaaaactc ttaaaacccg cctggcctgt gcataactgt ctggccagcg cacagccgaa 5160gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccc tacgccccgc cgcttcgcgt 5220cggcctatcg cggccgctgg ccgctcaaaa atggctggcc tacggccagg caatctacca 5280gggcgcggac aagccgcgcc gtcgccactc gaccgccggc gctgaggtct gcctcgtgaa 5340gaaggtgttg ctgactcata ccaggcctga atcgccccat catccagcca gaaagtgagg 5400gagccacggt tgatgagagc tttgttgtag gtggaccagt tggtgat ttt gaacttttgc 5460tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 5520aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 5580gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 5640atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5700gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 5760cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 5820gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc tctgcattaa 5880tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5940ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6000gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6060ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6120cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6180ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6240accctgccgc ttaccggata cctgtcc ttt gaacttttgc 5460tttgccacgg aacggtctgc gttgtcggga agatgcgtga tctgatcctt caactcagca 5520aaagttcgat ttattcaaca aagccgccgt cccgtcaagt cagcgtaatg ctctgccagt 5580gttacaacca attaaccaat tctgattaga aaaactcatc gagcatcaaa tgaaactgca 5640atttattcat atcaggatta tcaataccat atttttgaaa aagccgtttc tgtaatgaag 5700gagaaaactc accgaggcag ttccatagga tggcaagatc ctggtatcgg tctgcgattc 5760cgactcgtcc aacatcaata caacctatta atttcccctc gtcaaaaata aggttatcaa 5820gtgagaaatc accatgagtg acgactgaat ccggtgagaa tggcaaaagc tctgcattaa 5880tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 5940ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 6000gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 6060ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 6120cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 6180ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 6240accctgccgc ttaccggata cctgtcc gcc tttctccctt cgggaagcgt ggcgctttct 6300catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6360gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6420tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6480 gcc tttctccctt cgggaagcgt ggcgctttct 6300catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 6360gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 6420tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 6480

agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 6540actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6600gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6660aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6720gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6780aaaaggatct tcacctagat ccttttgatc cggaattaat tcctgtggtt ggcatgcaca 6840tacaaatgga cgaacggata aaccttttca cgccctttta aatatccgat tattctaata 6900aacgctcttt tctcttaggt ttacccgcca atatatcctg tcaaacactg atagtttaaa 6960ctgaaggcgg gaaacgacaa tctgatcatg agcggagaat taagggagtc acgttatgac 7020ccccgccgat gacgcgggac aagccgtttt acgtttggaa ctgacagaac cgcaacgctg 7080caggaattgg ccgcagcggc catttaaatc aattgggcgc gtacgtagca ctagtgcgcg 7140atcgcttaat taagcggcgc gcctaaagct tctggcagac aaagtggcag acatactgtc 7200ccacaaatga agatggaatc tgtaaaagaa aacgcgtgaa ataatgcgtc tgacaaaggt 7260taggtcggct gcctttaatc aataccaaag tggtccctac cacgatg agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac atatatcctg tcaaacactg atagtttaaa 6540actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 6600gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 6660aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 6720gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 6780aaaaggatct tcacctagat ccttttgatc cggaattaat tcctgtggtt ggcatgcaca 6840tacaaatgga cgaacggata aaccttttca cgccctttta aatatccgat tattctaata 6900aacgctcttt tctcttaggt ttacccgcca 6960ctgaaggcgg gaaacgacaa tctgatcatg agcggagaat taagggagtc acgttatgac 7020ccccgccgat gacgcgggac aagccgtttt acgtttggaa ctgacagaac cgcaacgctg 7080caggaattgg ccgcagcggc catttaaatc aattgggcgc gtacgtagca ctagtgcgcg 7140atcgcttaat taagcggcgc gcctaaagct tctggcagac aaagtggcag acatactgtc 7200ccacaaatga agatggaatc tgtaaaagaa aacgcgtgaa ataatgcgtc tgacaaaggt 7260taggtcggct gcctttaatc aataccaaag tggtccctac cacgatg gaa aaactgtgca 7320gtcggtttgg ctttttctga cgaacaaata agattcgtgg ccgacaggtg ggggtccacc 7380atgtgaaggc atcttcagac tccaataatg gagcaatgac gtaagggctt acgaaataag 7440taagggtagt ttgggaaatg tccactcacc cgtcagtcta taaatactta gcccctccct 7500cattgttaag ggagcaagga tccaccggtc gccaccatgg ccctgtccaa caagttcatc 7560ggcgacgaca tgaagatgac ctaccacatg gacggctgcg tgaacggcca ctacttcacc 7620gtgaagggcg agggcagcgg caagccctac gagggcaccc agacctccac cttcaaggtg 7680accatggcca acggcggccc cctggccttc tccttcgaca tcctgtccac cgtgttcatg 7740tacggcaacc gctgcttcac cgcctacccc accagcatgc ccgactactt caagcaggcc 7800ttccccgacg gcatgtccta cgagagaacc ttcacctacg aggacggcgg cgtggccacc 7860gccagctggg agatcagcct gaagggcaac tgcttcgagc acaagtccac cttccacggc 7920gtgaacttcc ccgccgacgg ccccgtgatg gccaagaaga ccaccggctg ggacccctcc 7980ttcgagaaga tgaccgtgtg cgacggcatc ttgaagggcg acgtgaccgc cttcctgatg 8040ctgcagggcg gcggcaacta cagatgccag ttccacacct cctacaagac caagaagccc 8100gtgaccatgc cccccaacca cgtggtg gaa aaactgtgca 7320gtcggtttgg ctttttctga cgaacaaata agattcgtgg ccgacaggtg ggggtccacc 7380atgtgaaggc atcttcagac tccaataatg gagcaatgac gtaagggctt acgaaataag 7440taagggtagt ttgggaaatg tccactcacc cgtcagtcta taaatactta gcccctccct 7500cattgttaag ggagcaagga tccaccggtc gccaccatgg ccctgtccaa caagttcatc 7560ggcgacgaca tgaagatgac ctaccacatg gacggctgcg tgaacggcca ctacttcacc 7620gtgaagggcg agggcagcgg caagccctac gagggcaccc agacctccac cttcaaggtg 7680accatggcca acggcggccc cctggccttc tccttcgaca tcctgtccac cgtgttcatg 7740tacggcaacc gctgcttcac cgcctacccc accagcatgc ccgactactt caagcaggcc 7800ttccccgacg gcatgtccta cgagagaacc ttcacctacg aggacggcgg cgtggccacc 7860gccagctggg agatcagcct gaagggcaac tgcttcgagc acaagtccac cttccacggc 7920gtgaacttcc ccgccgacgg ccccgtgatg gccaagaaga ccaccggctg ggacccctcc 7980ttcgagaaga tgaccgtgtg cgacggcatc ttgaagggcg acgtgaccgc cttcctgatg 8040ctgcagggcg gcggcaacta cagatgccag ttccacacct cctacaagac caagaagccc 8100gtgaccatgc cccccaacca cgtggtg gag caccgcatcg ccagaaccga cctggacaag 8160ggcggcaaca gcgtgcagct gaccgagcac gccgtggccc acatcacctc cgtggtgccc 8220ttctgagagc tctagatccc cgaatttccc cgatcgttca aacatttggc aataaagttt 8280cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 8340cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 8400 gag caccgcatcg ccagaaccga cctggacaag 8160ggcggcaaca gcgtgcagct gaccgagcac gccgtggccc acatcacctc cgtggtgccc 8220ttctgagagc tctagatccc cgaatttccc cgatcgttca aacatttggc aataaagttt 8280cttaagattg aatcctgttg ccggtcttgc gatgattatc atataatttc tgttgaatta 8340cgttaagcat gtaataatta acatgtaatg catgacgtta tttatgagat gggtttttat 8400

gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 8460ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgggtaccga 8520attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 8580atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 8640atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 8700tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 8760ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 8820gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 8880tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 8940gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9000ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9060atccgctcat gagacaataa ccctgataaa tgcttcaatg gcgcgccggt accagcttgc 9120atgcctgcag gtcgactcta gaggatcctg gcagacaaag tggcagacat actgtcccac 9180aaatgaagat ggaatctgta aaagaaaacg cgtgaaataa tgcgtct gattagagtc ccgcaattat acatttaata cgcgatagaa aacaaaatat agcgcgcaaa 8460ctaggataaa ttatcgcgcg cggtgtcatc tatgttacta gatcgggaat tgggtaccga 8520attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta 8580atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg 8640atcgcccttc ccaacagttg cgcagcctga atggcgaatg gcgcctgatg cggtattttc 8700tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 8760ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 8820gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 8880tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 8940gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 9000ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 9060atccgctcat gagacaataa ccctgataaa tgcttcaatg gcgcgccggt accagcttgc 9120atgcctgcag gtcgactcta gaggatcctg gcagacaaag tggcagacat actgtcccac 9180aaatgaagat ggaatctgta aaagaaaacg cgtgaaataa tgcgtct gac aaaggttagg 9240tcggctgcct ttaatcaata ccaaagtggt ccctaccacg atggaaaaac tgtgcagtcg 9300gtttggcttt ttctgacgaa caaataagat tcgtggccga caggtggggg tccaccatgt 9360gaaggcatct tcagactcca ataatggagc aatgacgtaa gggcttacga aataagtaag 9420ggtagtttgg gaaatgtcca ctcacccgtc agtctataaa tacttagccc ctccctcatt 9480gttaagggag caaaatctca gagagatagt cctagagaga gaaagagagc aagtagccta 9540gaagta 9546<210>3<211>10604<212>DNA<213>人工<220> gac aaaggttagg 9240tcggctgcct ttaatcaata ccaaagtggt ccctaccacg atggaaaaac tgtgcagtcg 9300gtttggcttt ttctgacgaa caaataagat tcgtggccga caggtggggg tccaccatgt 9360gaaggcatct tcagactcca ataatggagc aatgacgtaa gggcttacga aataagtaag 9420ggtagtttgg gaaatgtcca ctcacccgtc agtctataaa tacttagccc ctccctcatt 9480gttaagggag caaaatctca gagagatagt cctagagaga gaaagagagc aagtagccta 9540gaagta 9546 & lt; 210 & gt; 3 & lt; 211 & gt; 10604 & lt; 212 & gt; DNA & lt; 213 & gt; Artificial & lt; 220 & gt;

<223>pBSC11234<400>3cgcgcctaaa gcttgcatgc ctgcaggtcg actctagagg atcctggcag acaaagtggc 60agacatactg tcccacaaat gaagatggaa tctgtaaaag aaaacgcgtg aaataatgcg 120tctgacaaag gttaggtcgg ctgcctttaa tcaataccaa agtggtccct accacgatgg 180aaaaactgtg cagtcggttt ggctttttct gacgaacaaa taagattcgt ggccgacagg 240tgggggtcca ccatgtgaag gcatcttcag actccaataa tggagcaatg acgtaagggc 300ttacgaaata agtaagggta gtttgggaaa tgtccactca cccgtcagtc tataaatact 360 cccgtcagtc tataaatact 3cgcgcctaaa gcttgcatgc ctgcaggtcg actctagagg atcctggcag acaaagtggc 60agacatactg tcccacaaat gaagatggaa tctgtaaaag aaaacgcgtg aaataatgcg 120tctgacaaag gttaggtcgg ctgcctttaa tcaataccaa agtggtccct accacgatgg 180aaaaactgtg cagtcggttt ggctttttct gacgaacaaa taagattcgt ggccgacagg 240tgggggtcca ccatgtgaag gcatcttcag actccaataa tggagcaatg acgtaagggc 300ttacgaaata agtaagggta gtttgggaaa tgtccactca 360; & lt; 223 & gt; pBSC11234 & lt; 400 & gt

tagcccctcc ctcattgtta agggagcaaa atctcagaga gatagtccta gagagagaaa 420gagagcaagt agcctagaag taggatcccc gatcatgcaa aaactcatta actcagtgca 480aaactatgcc tggggcagca aaacggcgtt gactgaactt tatggtatgg aaaatccgtc 540cagccagccg atggccgagc tgtggatggg cgcacatccg aaaagcagtt cacgagtgca 600gaatgccgcc ggagatatcg tttcactgcg tgatgtgatt gagagtgata aatcgactct 660gctcggagag gccgttgcca aacgctttgg cgaactgcct ttcctgttca aagtattatg 720cgcagcacag ccactctcca ttcaggttca tccaaacaaa cacaattctg aaatcggttt 780tgccaaagaa aatgccgcag gtatcccgat ggatgccgcc gagcgtaact ataaagatcc 840taaccacaag ccggagctgg tttttgcgct gacgcctttc cttgcgatga acgcgtttcg 900tgaattttcc gagattgtct ccctactcca gccggtcgca ggtgcacatc cggcgattgc 960tcacttttta caacagcctg atgccgaacg tttaagcgaa ctgttcgcca gcctgttgaa 1020tatgcagggt gaagaaaaat cccgcgcgct ggcgatttta aaatcggccc tcgatagcca 1080gcagggtgaa ccgtggcaaa cgattcgttt aatttctgaa ttttacccgg aagacagcgg 1140tctgttctcc ccgctattgc tgaatgtggt gaaattgaac cctggcg tagcccctcc ctcattgtta agggagcaaa atctcagaga gatagtccta gagagagaaa 420gagagcaagt agcctagaag taggatcccc gatcatgcaa aaactcatta actcagtgca 480aaactatgcc tggggcagca aaacggcgtt gactgaactt tatggtatgg aaaatccgtc 540cagccagccg atggccgagc tgtggatggg cgcacatccg aaaagcagtt cacgagtgca 600gaatgccgcc ggagatatcg tttcactgcg tgatgtgatt gagagtgata aatcgactct 660gctcggagag gccgttgcca aacgctttgg cgaactgcct ttcctgttca aagtattatg 720cgcagcacag ccactctcca ttcaggttca tccaaacaaa cacaattctg aaatcggttt 780tgccaaagaa aatgccgcag gtatcccgat ggatgccgcc gagcgtaact ataaagatcc 840taaccacaag ccggagctgg tttttgcgct gacgcctttc cttgcgatga acgcgtttcg 900tgaattttcc gagattgtct ccctactcca gccggtcgca ggtgcacatc cggcgattgc 960tcacttttta caacagcctg atgccgaacg tttaagcgaa ctgttcgcca gcctgttgaa 1020tatgcagggt gaagaaaaat cccgcgcgct ggcgatttta aaatcggccc tcgatagcca 1080gcagggtgaa ccgtggcaaa cgattcgttt aatttctgaa ttttacccgg aagacagcgg 1140tctgttctcc ccgctattgc tgaatgtggt gaaattgaac cctggcg aag cgatgttcct 1200gttcgctgaa acaccgcacg cttacctgca aggcgtggcg ctggaagtga tggcaaactc 1260cgataacgtg ctgcgtgcgg gtctgacgcc taaatacatt gatattccgg aactggttgc 1320caatgtgaaa ttcgaagcca aaccggctaa ccagttgttg acccagccgg tgaaacaagg 1380tgcagaactg gacttcccga ttccagtgga tgattttgcc ttctcgctgc atgaccttag 1440tgataaagaa accaccatta gccagcagag tgccgccatt ttgttctgcg tcgaaggcga 1500tgcaacgttg tggaaaggtt ctcagcagtt acagcttaaa ccgggtgaat cagcgtttat 1560tgccgccaac gaatcaccgg tgactgtcaa aggccacggc cgtttagcgc gtgtttacaa 1620caagctgtaa gagcttactg aaaaaattaa catctcttgc taagctggga gctctagatc 1680cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 1740tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 1800taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 1860atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 1920cgcggtgtca tctatgttac tagatcggga attgggtacc atgcccgggc ggccagcatg 1980gccgtatccg caatgtgtta ttaagtt aag cgatgttcct 1200gttcgctgaa acaccgcacg cttacctgca aggcgtggcg ctggaagtga tggcaaactc 1260cgataacgtg ctgcgtgcgg gtctgacgcc taaatacatt gatattccgg aactggttgc 1320caatgtgaaa ttcgaagcca aaccggctaa ccagttgttg acccagccgg tgaaacaagg 1380tgcagaactg gacttcccga ttccagtgga tgattttgcc ttctcgctgc atgaccttag 1440tgataaagaa accaccatta gccagcagag tgccgccatt ttgttctgcg tcgaaggcga 1500tgcaacgttg tggaaaggtt ctcagcagtt acagcttaaa ccgggtgaat cagcgtttat 1560tgccgccaac gaatcaccgg tgactgtcaa aggccacggc cgtttagcgc gtgtttacaa 1620caagctgtaa gagcttactg aaaaaattaa catctcttgc taagctggga gctctagatc 1680cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 1740tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 1800taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 1860atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 1920cgcggtgtca tctatgttac tagatcggga attgggtacc atgcccgggc ggccagcatg 1980gccgtatccg caatgtgtta ttaagtt gtc taagcgtcaa tttgtttaca ccacaatata 2040tcctgccacc agccagccaa cagctccccg accggcagct cggcacaaaa tcaccactcg 2100atacaggcag cccatcagaa ttaattctca tgtttgacag cttatcatcg actgcacggt 2160gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt 2220aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc 2280 gtc taagcgtcaa tttgtttaca ccacaatata 2040tcctgccacc agccagccaa cagctccccg accggcagct cggcacaaaa tcaccactcg 2100atacaggcag cccatcagaa ttaattctca tgtttgacag cttatcatcg actgcacggt 2160gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt 2220aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc 2280

gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc 2340ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagaccatg 2400agggaagcgt tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag 2460cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc 2520ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca 2580acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag 2640attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat 2700ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc 2760ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat 2820agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat 2880ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc 2940gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa 3000atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag 3060cccgtcatac ttgaagctag gcaggcttat cttggacaag aagatcg gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc 2340ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagaccatg 2400agggaagcgt tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag 2460cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc 2520ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca 2580acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag 2640attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat 2700ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc 2760ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat 2820agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat 2880ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc 2940gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa 3000atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag 3060cccgtcatac ttgaagctag gcaggcttat cttggacaag aagatcg ctt ggcctcgcgc 3120gcagatcagt tggaagaatt tgttcactac gtgaaaggcg agatcaccaa agtagtcggc 3180aaataaagct ctagtggatc tccgtacccc cgggggatct ggctcgcggc ggacgcacga 3240cgccggggcg agaccatagg cgatctccta aatcaatagt agctgtaacc tcgaagcgtt 3300tcacttgtaa caacgattga gaatttttgt cataaaattg aaatacttgg ttcgcatttt 3360tgtcatccgc ggtcagccgc aattctgacg aactgcccat ttagctggag atgattgtac 3420atccttcacg tgaaaatttc tcaagcgctg tgaacaaggg ttcagatttt agattgaaag 3480gtgagccgtt gaaacacgtt cttcttgtcg atgacgacgt cgctatgcgg catcttatta 3540ttgaatacct tacgatccac gccttcaaag tgaccgcggt agccgacagc acccagttca 3600caagagtact ctcttccgcg acggtcgatg tcgtggttgt tgatctaaat ttaggtcgtg 3660aagatgggct cgagatcgtt cgtaatctgg cggcaaagtc tgatattcca atcataatta 3720tcagtggcga ccgccttgag gagacggata aagttgttgc actcgagcta ggagcaagtg 3780attttatcgc taagccgttc agtatcagag agtttctagc acgcattcgg gttgccttgc 3840gcgtgcgccc caacgttgtc cgctccaaag accgacggtc tttttgtttt actgactgga 3900cacttaatct caggcaacgt cgcttga ctt ggcctcgcgc 3120gcagatcagt tggaagaatt tgttcactac gtgaaaggcg agatcaccaa agtagtcggc 3180aaataaagct ctagtggatc tccgtacccc cgggggatct ggctcgcggc ggacgcacga 3240cgccggggcg agaccatagg cgatctccta aatcaatagt agctgtaacc tcgaagcgtt 3300tcacttgtaa caacgattga gaatttttgt cataaaattg aaatacttgg ttcgcatttt 3360tgtcatccgc ggtcagccgc aattctgacg aactgcccat ttagctggag atgattgtac 3420atccttcacg tgaaaatttc tcaagcgctg tgaacaaggg ttcagatttt agattgaaag 3480gtgagccgtt gaaacacgtt cttcttgtcg atgacgacgt cgctatgcgg catcttatta 3540ttgaatacct tacgatccac gccttcaaag tgaccgcggt agccgacagc acccagttca 3600caagagtact ctcttccgcg acggtcgatg tcgtggttgt tgatctaaat ttaggtcgtg 3660aagatgggct cgagatcgtt cgtaatctgg cggcaaagtc tgatattcca atcataatta 3720tcagtggcga ccgccttgag gagacggata aagttgttgc actcgagcta ggagcaagtg 3780attttatcgc taagccgttc agtatcagag agtttctagc acgcattcgg gttgccttgc 3840gcgtgcgccc caacgttgtc cgctccaaag accgacggtc tttttgtttt actgactgga 3900cacttaatct caggcaacgt cgcttga tgt ccgaagctgg cggtgaggtg aaacttacgg 3960caggtgagtt caatcttctc ctcgcgtttt tagagaaacc ccgcgacgtt ctatcgcgcg 4020agcaacttct cattgccagt cgagtacgcg acgaggaggt ttatgacagg agtatagatg 4080ttctcatttt gaggctgcgc cgcaaacttg aggcagatcc gtcaagccct caactgataa 4140 tgt ccgaagctgg cggtgaggtg aaacttacgg 3960caggtgagtt caatcttctc ctcgcgtttt tagagaaacc ccgcgacgtt ctatcgcgcg 4020agcaacttct cattgccagt cgagtacgcg acgaggaggt ttatgacagg agtatagatg 4080ttctcatttt gaggctgcgc cgcaaacttg aggcagatcc gtcaagccct caactgataa 4140

aaacagcaag aggtgccggt tatttctttg acgcggacgt gcaggtttcg cacgggggga 4200cgatggcagc ctgagccaat tcccagatcc ccgaggaatc ggcgtgagcg gtcgcaaacc 4260atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 4320cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 4380agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 4440gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 4500cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 4560tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 4620cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 4680ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 4740gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 4800gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 4860tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 4920ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcg aaacagcaag aggtgccggt tatttctttg acgcggacgt gcaggtttcg cacgggggga 4200cgatggcagc ctgagccaat tcccagatcc ccgaggaatc ggcgtgagcg gtcgcaaacc 4260atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 4320cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 4380agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 4440gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 4500cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 4560tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 4620cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 4680ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 4740gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 4800gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 4860tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 4920ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcg aga tcgagctagc 4980tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 5040cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 5100cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 5160cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 5220ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 5280caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 5340aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 5400tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 5460cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 5520cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 5580gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 5640cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 5700cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 5760gcgctgaggt ctgcctcgtg aagaagg aga tcgagctagc 4980tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 5040cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 5100cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 5160cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 5220ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 5280caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 5340aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 5400tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 5460cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 5520cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 5580gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 5640cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 5700cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 5760gcgctgaggt ctgcctcgtg aagaagg tgt tgctgactca taccaggcct gaatcgcccc 5820atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca 5880gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt 5940gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa 6000gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca 6060 tgt tgctgactca taccaggcct gaatcgcccc 5820atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca 5880gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt 5940gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa 6000gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca 6060

tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 6120aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 6180tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 6240tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 6300aatggcaaaa gctctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 6360tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 6420agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 6480aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 6540gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 6600tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 6660cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 6720ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 6780cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 6840atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttat tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga aaaggccagg aaccgtaaaa aggccgcgtt 6120aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 6180tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 6240tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 6300aatggcaaaa gctctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 6360tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 6420agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 6480aggaaagaac atgtgagcaa aaggccagca 6540gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 6600tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 6660cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 6720ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 6780cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 6840atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttat cgc cactggcagc 6900agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 6960gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 7020gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 7080tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 7140agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 7200gattttggtc atgagattat caaaaaggat cttcacctag atccttttga tccggaatta 7260attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 7320taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 7380tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 7440attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 7500aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccatttaaa tcaattgggc 7560gcgtacgtag cactagtgcg cgatcgctta attaagcggc gcgcctgcag gcggccgcac 7620aattattata tcaaaatggc aaaaacattt aatacgtatt atttaagaaa aaaatatgta 7680ataatatatt tatattttaa tatctat cgc cactggcagc 6900agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 6960gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 7020gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 7080tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 7140agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 7200gattttggtc atgagattat caaaaaggat cttcacctag atccttttga tccggaatta 7260attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 7320taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 7380tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 7440attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 7500aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccatttaaa tcaattgggc 7560gcgtacgtag cactagtgcg cgatcgctta attaagcggc gcgcctgcag gcggccgcac 7620aattattata tcaaaatggc aaaaacattt aatacgtatt atttaagaaa aaaatatgta 7680ataatatatt tatattttaa tatctat tct tatgtatttt ttaaaaatct attatatatt 7740gatcaactaa aatattttta tatctacact tattttgcat ttttatcaat tttcttgcgt 7800tttttggcat atttaataat gactattctt taataatcga tcattattct tacatggtac 7860atattgttgg aaccatatga agtgtccatt gcatttgact atgtggatag tgttttgatc 7920 tct ttaaaaatct attatatatt 7740gatcaactaa aatattttta tatctacact tattttgcat ttttatcaat tttcttgcgt 7800tttttggcat atttaataat gactattctt taataatcga tcattattct tacatggtac 7860atattgttgg aaccatatga agtgtccatt gcatttgact atgtggatag tatgtatttt tgttttgatc 7920

caggcctcca tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt 7980atccttcctc catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc 8040ttggatcata agaaaaagcc aaggaacaaa agaagacaaa acacaatggg agtatccttt 8100gcatagcaat gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat 8160cacttatcca ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag 8220ccatgcacaa caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa 8280ctcaacccat catgagccca cacatttgtt gtttctaacc caacctcaaa ctcgtattct 8340cttccgccac ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc 8400caaatgtcca tgcatgttaa caagacctat gactataaat atctgcaatc tcggcccagg 8460ttttcatcat caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat 8520actccaccgg atccaccatg gccaagctag ttttttccct ttgttttctg cttttcagtg 8580gctgctgctt cgctgagatt ttcggcaaga ccttccgcga gggccgcttc gtgctcaagg 8640agaagaactt caccgtggag ttcgccgtgg agaagatcca cctcggctgg aagatatcgg 8700gccgcgtgaa gggctcgccg ggccgcctcg aggtgctccg caccaag caggcctcca tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt 7980atccttcctc catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc 8040ttggatcata agaaaaagcc aaggaacaaa agaagacaaa acacaatggg agtatccttt 8100gcatagcaat gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat 8160cacttatcca ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag 8220ccatgcacaa caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa 8280ctcaacccat catgagccca cacatttgtt gtttctaacc caacctcaaa ctcgtattct 8340cttccgccac ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc 8400caaatgtcca tgcatgttaa caagacctat gactataaat atctgcaatc tcggcccagg 8460ttttcatcat caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat 8520actccaccgg atccaccatg gccaagctag ttttttccct ttgttttctg cttttcagtg 8580gctgctgctt cgctgagatt ttcggcaaga ccttccgcga gggccgcttc gtgctcaagg 8640agaagaactt caccgtggag ttcgccgtgg agaagatcca cctcggctgg aagatatcgg 8700gccgcgtgaa gggctcgccg ggccgcctcg aggtgctccg caccaag gcc ccggagaagg 8760tgctcgtgaa caactggcag tcctggggcc cgtgccgcgt ggtggacgcc ttctccttca 8820agccgccgga gatcgacccg aactggcgct acaccgcatc cgtggtgccg gacgtgctcg 8880agcgcaacct gcagtccgac tacttcgtgg ccgaggaggg caaggtgtac ggcttcctct 8940cctccaagat cgcccacccg ttcttcgcgg tggaggacgg cgagctggtg gcctacctcg 9000agtacttcga cgtggagttc gacgacttcg tgccgctgga gccgctcgtg gtgctcgagg 9060acccgaacac cccgctcctc ctcgagaagt acgccgagct ggtgggcatg gagaacaacg 9120cccgggtgcc gaagcacacg ccgaccggct ggtgctcctg gtatcactac ttcctcgacc 9180tcacctggga ggagaccctc aagaacctca agctcgccaa gaacttcccg ttcgaggtgt 9240tccagatcga cgacgcctac gagaaggaca tcggcgactg gctcgtgacc cgcggcgact 9300tcccgtccgt ggaggagatg gccaaggtga tcgccgagaa cggcttcatc cccggcatct 9360ggaccgcccc gttctccgtg tccgagacta gtgacgtgtt caacgagcac ccggactggg 9420tggtgaagga gaacggcgag ccgaagatgg cctaccgcaa ctggaacaag aagatttacg 9480ccctcgacct ctccaaggac gaggtgctca actggctctt cgacctcttc tcctccctcc 9540gcaagatggg ctaccgctac ttcaaga gcc ccggagaagg 8760tgctcgtgaa caactggcag tcctggggcc 8820agccgccgga cgtgccgcgt ggtggacgcc ttctccttca ggagaccctc aagaacctca agctcgccaa gatcgacccg aactggcgct acaccgcatc cgtggtgccg gacgtgctcg 8880agcgcaacct gcagtccgac tacttcgtgg ccgaggaggg caaggtgtac ggcttcctct 8940cctccaagat cgcccacccg ttcttcgcgg tggaggacgg cgagctggtg gcctacctcg 9000agtacttcga cgtggagttc gacgacttcg tgccgctgga gccgctcgtg gtgctcgagg 9060acccgaacac cccgctcctc ctcgagaagt acgccgagct ggtgggcatg gagaacaacg 9120cccgggtgcc gaagcacacg ccgaccggct ggtgctcctg gtatcactac ttcctcgacc 9180tcacctggga gaacttcccg ttcgaggtgt 9240tccagatcga cgacgcctac gagaaggaca tcggcgactg gctcgtgacc cgcggcgact 9300tcccgtccgt ggaggagatg gccaaggtga tcgccgagaa cggcttcatc cccggcatct 9360ggaccgcccc gttctccgtg tccgagacta gtgacgtgtt caacgagcac ccggactggg 9420tggtgaagga gaacggcgag ccgaagatgg cctaccgcaa ctggaacaag aagatttacg 9480ccctcgacct ctccaaggac gaggtgctca actggctctt cgacctcttc tcctccctcc 9540gcaagatggg ctaccgctac ttcaaga tcg acttcctctt cgcgggcgcc gtgccggggg 9600agcgcaagaa gaacatcacc ccgatccagg ccttccgcaa gggcatcgag accatccgca 9660aggccgtggg ggaggactcc ttcatcctcg gctgcggctc ccccctcctc ccggccgtgg 9720gctgcgtgga tggcatgcgc atcggcccgg acaccgcccc gttctgggga gagcacatcg 9780aggacaacgg cgccccggcg gcccgctggg ccctccgcaa cgccatcacc cgctacttca 9840 tcg acttcctctt cgcgggcgcc gtgccggggg 9600agcgcaagaa gaacatcacc ccgatccagg ccttccgcaa gggcatcgag accatccgca 9660aggccgtggg ggaggactcc ttcatcctcg gctgcggctc ccccctcctc ccggccgtgg 9720gctgcgtgga tggcatgcgc atcggcccgg acaccgcccc gttctgggga gagcacatcg 9780aggacaacgg cgccccggcg gcccgctggg ccctccgcaa cgccatcacc cgctacttca 9840

tgcacgaccg cttctggctc aacgacccgg actgcctcat cctccgcgag gagaagaccg 9900acctcaccca gaaggagaag gagctgtact cctacacctg cggcgttcta gacaacatga 9960tcatcgagtc cgacgacctc tccctcgtgc gcgaccacgg caagaaggtg ctcaaggaga 10020ccctcgagct gctcgggggc aggccgcgcg tgcagaacat catgtccgag gacctccgct 10080acgagatcgt gtcctcgggc accctctccg gcaacgtgaa gatcgtggtg gacctcaact 10140cccgcgagta ccacctcgag aaggagggca agtcctccct caagaagcgc gtggtgaagc 10200gggaggacgg caggaacttc tacttctacg aggagggcga gcgcgagtga aagcttgacg 10260tcactagtgc gatcgcgcta gccatggccg gcctaggcgc ccgggagatc cccgaatttc 10320cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 10380gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 10440tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 10500tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 10560tctatgttac tagatcggga attcctcgag tctagacctg cagg 10604<210>4<211>8757<212>DNA<213>人工<22 tgcacgaccg cttctggctc aacgacccgg actgcctcat cctccgcgag gagaagaccg 9900acctcaccca gaaggagaag gagctgtact cctacacctg cggcgttcta gacaacatga 9960tcatcgagtc cgacgacctc tccctcgtgc gcgaccacgg caagaaggtg ctcaaggaga 10020ccctcgagct gctcgggggc aggccgcgcg tgcagaacat catgtccgag gacctccgct 10080acgagatcgt gtcctcgggc accctctccg gcaacgtgaa gatcgtggtg gacctcaact 10140cccgcgagta ccacctcgag aaggagggca agtcctccct caagaagcgc gtggtgaagc 10200gggaggacgg caggaacttc tacttctacg aggagggcga gcgcgagtga aagcttgacg 10260tcactagtgc gatcgcgcta gccatggccg gcctaggcgc ccgggagatc cccgaatttc 10320cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 10380gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 10440tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 10500tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 10560tctatgttac tagatcggga attcctcgag tctagacctg cagg 10604 & lt; 210 & gt; 4 & lt; 211 & gt; 8757 & lt; 212 & gt; DNA & lt; 213 & gt; artificial & lt; 22 0> 0 & gt;

<223>pBSC11369<400>4aagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa 60aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac 120caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac 180aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcatctt cagactccaa 240taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac 300tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccac 360cggtcgccac catggcccag tccaagcacg gcctgaccaa ggagatgacc atgaagtacc 420gcatggaggg ctgcgtggac ggccacaagt tcgtgatcac cggcgagggc atcggctacc 480ccttcaaggg caagcaggcc atcaacctgt gcgtggtgga gggcggcccc ttgcccttcg 540ccgaggacat cttgtccgcc gccttcatgt acggcaaccg cgtgttcacc gagtaccccc 600aggacatcgt cgactacttc aagaactcct gccccgccgg ctacacctgg gaccgctcct 660tcctgttcga ggacggcgcc gtgtgcatct gcaacgccga catcaccgtg agcgtggagg 720agaactgcat gtaccacgag tccaagttct acggcgtgaa cttccccgcc gacggccccg 780 & Lt; 223 & gt; pBSC11369 & lt; 400 & gt; 4aagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa 60aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac 120caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac 180aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcatctt cagactccaa 240taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac 300tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccac 360cggtcgccac catggcccag tccaagcacg gcctgaccaa ggagatgacc atgaagtacc 420gcatggaggg ctgcgtggac ggccacaagt tcgtgatcac cggcgagggc atcggctacc 480ccttcaaggg caagcaggcc atcaacctgt gcgtggtgga gggcggcccc ttgcccttcg 540ccgaggacat cttgtccgcc gccttcatgt acggcaaccg cgtgttcacc gagtaccccc 600aggacatcgt cgactacttc aagaactcct gccccgccgg ctacacctgg gaccgctcct 660tcctgttcga ggacggcgcc gtgtgcatct gcaacgccga catcaccgtg agcgtggagg 720agaactgcat gtaccacgag tccaagttct acggcgtgaa cttccccgcc gacggccccg 780

tgatgaagaa gatgaccgac aactgggagc cctcctgcga gaagatcatc cccgtgccca 840agcagggcat cttgaagggc gacgtgagca tgtacctgct gctgaaggac ggtggccgct 900tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgt gccccgcaag atgcccgact 960ggcacttcat ccagcacaag ctgacccgcg aggaccgcag cgacgccaag aaccagaagt 1020ggcacctgac cgagcacgcc atcgcctccg gctccgcctt gccctgctct agatcccgaa 1080tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 1140tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 1200gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 1260ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 1320gtcatctatg ttactagatc gggaattggg gaaatttacc ggtgccgaat ttccccgatc 1380cagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa 1440aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac 1500caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac 1560aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcat tgatgaagaa gatgaccgac aactgggagc cctcctgcga gaagatcatc cccgtgccca 840agcagggcat cttgaagggc gacgtgagca tgtacctgct gctgaaggac ggtggccgct 900tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgt gccccgcaag atgcccgact 960ggcacttcat ccagcacaag ctgacccgcg aggaccgcag cgacgccaag aaccagaagt 1020ggcacctgac cgagcacgcc atcgcctccg gctccgcctt gccctgctct agatcccgaa 1080tttccccgat cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg 1140tcttgcgatg attatcatat aatttctgtt gaattacgtt aagcatgtaa taattaacat 1200gtaatgcatg acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat 1260ttaatacgcg atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt 1320gtcatctatg ttactagatc gggaattggg gaaatttacc ggtgccgaat ttccccgatc 1380cagcttctgg cagacaaagt ggcagacata ctgtcccaca aatgaagatg gaatctgtaa 1440aagaaaacgc gtgaaataat gcgtctgaca aaggttaggt cggctgcctt taatcaatac 1500caaagtggtc cctaccacga tggaaaaact gtgcagtcgg tttggctttt tctgacgaac 1560aaataagatt cgtggccgac aggtgggggt ccaccatgtg aaggcat ctt cagactccaa 1620taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac 1680tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccat 1740gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 1800cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 1860aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 1920ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 1980ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 2040agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 2100gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 2160cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 2220ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 2280gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 2340caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 2400gttcggggat tcccaatacg aggtcgc ctt cagactccaa 1620taatggagca atgacgtaag ggcttacgaa ataagtaagg gtagtttggg aaatgtccac 1680tcacccgtca gtctataaat acttagcccc tccctcattg ttaagggagc aaggatccat 1740gaaaaagcct gaactcaccg cgacgtctgt cgagaagttt ctgatcgaaa agttcgacag 1800cgtctccgac ctgatgcagc tctcggaggg cgaagaatct cgtgctttca gcttcgatgt 1860aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgcc gatggtttct acaaagatcg 1920ttatgtttat cggcactttg catcggccgc gctcccgatt ccggaagtgc ttgacattgg 1980ggaattcagc gagagcctga cctattgcat ctcccgccgt gcacagggtg tcacgttgca 2040agacctgcct gaaaccgaac tgcccgctgt tctgcagccg gtcgcggagg ccatggatgc 2100gatcgctgcg gccgatctta gccagacgag cgggttcggc ccattcggac cgcaaggaat 2160cggtcaatac actacatggc gtgatttcat atgcgcgatt gctgatcccc atgtgtatca 2220ctggcaaact gtgatggacg acaccgtcag tgcgtccgtc gcgcaggctc tcgatgagct 2280gatgctttgg gccgaggact gccccgaagt ccggcacctc gtgcacgcgg atttcggctc 2340caacaatgtc ctgacggaca atggccgcat aacagcggtc attgactgga gcgaggcgat 2400gttcggggat tcccaatacg aggtcgc caa catcttcttc tggaggccgt ggttggcttg 2460tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 2520gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 2580caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 2640cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 2700 caa catcttcttc tggaggccgt ggttggcttg 2460tatggagcag cagacgcgct acttcgagcg gaggcatccg gagcttgcag gatcgccgcg 2520gctccgggcg tatatgctcc gcattggtct tgaccaactc tatcagagct tggttgacgg 2580caatttcgat gatgcagctt gggcgcaggg tcgatgcgac gcaatcgtcc gatccggagc 2640cgggactgtc gggcgtacac aaatcgcccg cagaagcgcg gccgtctgga ccgatggctg 2700

tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 2760atagggatcc cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat 2820tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 2880atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 2940tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 3000aattatcgcg cgcggtgtca tctatgttac tagatcggga attagcggcc cgaattcact 3060ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 3120tgcagcacat ccccctttcg ccaggggcgg ccagcatggc cgtatccgca atgtgttatt 3180aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 3240gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagaatt 3300aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag 3360gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc 3420gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg 3480caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtat tgtagaagta ctcgccgata gtggaaaccg acgccccagc actcgtccga gggcaaagga 2760atagggatcc cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat 2820tgaatcctgt tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc 2880atgtaataat taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag 2940tcccgcaatt atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata 3000aattatcgcg cgcggtgtca tctatgttac tagatcggga attagcggcc cgaattcact 3060ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct 3120tgcagcacat ccccctttcg ccaggggcgg ccagcatggc cgtatccgca atgtgttatt 3180aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 3240gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagaatt 3300aattctcatg tttgacagct tatcatcgac tgcacggtgc accaatgctt ctggcgtcag 3360gcagccatcg gaagctgtgg tatggctgtg caggtcgtaa atcactgcat aattcgtgtc 3420gctcaaggcg cactcccgtt ctggataatg ttttttgcgc cgacatcata acggttctgg 3480caaatattct gaaatgagct gttgacaatt aatcatccgg ctcgtat aat gtgtggaatt 3540gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag 3600tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 3660tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 3720atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 3780accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 3840ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 3900ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 3960ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 4020cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 4080ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta 4140cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 4200ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 4260aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 4320ttcactacgt gaaaggcgag atcacca aat gtgtggaatt 3540gtgagcggat aacaatttca cacaggaaac agaccatgag ggaagcgttg atcgccgaag 3600tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa ccgacgttgc 3660tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac agtgatattg 3720atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct ttgatcaacg 3780accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct gtagaagtca 3840ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc gaactgcaat 3900ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc acgatcgaca 3960ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg gtaggtccag 4020cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg ctaaatgaaa 4080ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat gtagtgctta 4140cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag gatgtcgctg 4200ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt gaagctaggc 4260aggcttatct tggacaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg 4320ttcactacgt gaaaggcgag atcacca aag tagtcggcaa ataaagctct agtggatctc 4380cgtacccggg gatctggctc gcggcggacg cacgacgccg gggcgagacc ataggcgatc 4440tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt 4500tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca gccgcaattc 4560 aag tagtcggcaa ataaagctct agtggatctc 4380cgtacccggg gatctggctc gcggcggacg cacgacgccg gggcgagacc ataggcgatc 4440tcctaaatca atagtagctg taacctcgaa gcgtttcact tgtaacaacg attgagaatt 4500tttgtcataa aattgaaata cttggttcgc atttttgtca tccgcggtca gccgcaattc 4560

tgacgaactg cccatttagc tggagatgat tgtacatcct tcacgtgaaa atttctcaag 4620cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac acgttcttct 4680tgtcgatgac gacgtcgcta tgcggcatct tattattgaa taccttacga tccacgcctt 4740caaagtgacc gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt 4800cgatgtcgtg gttgttgatc tagatttagg tcgtgaagat gggctcgaga tcgttcgtaa 4860tctggcggca aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac 4920ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc cgttcagtat 4980cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc 5040caaagaccga cggtcttttt gttttactga ctggacactt aatctcaggc aacgtcgctt 5100gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc ttctcctcgc 5160gtttttagag aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt 5220acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc tgcgccgcaa 5280acttgaggca gatccgtcaa gccctcaact gataaaaaca gcaagaggtg ccggttattt 5340ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatg gcagcct tgacgaactg cccatttagc tggagatgat tgtacatcct atttctcaag 4620cgctgtgaac aagggttcag attttagatt gaaaggtgag ccgttgaaac acgttcttct 4680tgtcgatgac gacgtcgcta tgcggcatct tattattgaa taccttacga tccacgcctt 4740caaagtgacc gcggtagccg acagcaccca gttcacaaga gtactctctt ccgcgacggt 4800cgatgtcgtg gttgttgatc tagatttagg tcgtgaagat gggctcgaga tcgttcgtaa 4860tctggcggca aagtctgata ttccaatcat aattatcagt ggcgaccgcc ttgaggagac 4920ggataaagtt gttgcactcg agctaggagc aagtgatttt atcgctaagc cgttcagtat 4980cagagagttt ctagcacgca ttcgggttgc cttgcgcgtg cgccccaacg ttgtccgctc 5040caaagaccga cggtcttttt tcacgtgaaa gttttactga ctggacactt aatctcaggc aacgtcgctt 5100gatgtccgaa gctggcggtg aggtgaaact tacggcaggt gagttcaatc ttctcctcgc 5160gtttttagag aaaccccgcg acgttctatc gcgcgagcaa cttctcattg ccagtcgagt 5220acgcgacgag gaggtttatg acaggagtat agatgttctc attttgaggc tgcgccgcaa 5280acttgaggca gatccgtcaa gccctcaact gataaaaaca gcaagaggtg ccggttattt 5340ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatg gcagcct gag ccaattccca 5400gatccccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg 5460gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc 5520atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa 5580gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac 5640gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc 5700atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc 5760cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt 5820gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga 5880taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta 5940ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc 6000attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc 6060ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa 6120accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca 6180gaaggcaaga acccggacgt gctgacg gag ccaattccca 5400gatccccgag gaatcggcgt gagcggtcgc aaaccatccg gcccggtaca aatcggcgcg 5460gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca gcggcaacgc 5520atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg aatccgcaaa 5580gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc caagggcgac 5640gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga tagtcgcagc 5700atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg cgaggtgatc 5760cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg catggccagt 5820gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc catgaaccga 5880taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt tgcggacgta 5940ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt agaaacctgc 6000attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa gaacggccgc 6060ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt aaagagcgaa 6120accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg cgagatcaca 6180gaaggcaaga acccggacgt gctgacg gtt caccccgatt actttttgat cgatcccggc 6240atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg 6300ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc 6360accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg 6420gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc 6480 gtt caccccgatt actttttgat cgatcccggc 6240atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga agccagatgg 6300ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa gttctgtttc 6360accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa ggaggaggcg 6420gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg cgaagcatcc 6480

gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt 6540cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg 6600aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa 6660gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt 6720aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag 6780ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg 6840cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg 6900cgcggacaag ccgcgccgtc gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa 6960ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag 7020ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt 7080gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa 7140gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt 7200acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt 7260tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttc gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg ggaaaaaggt 6540cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc gtacattggg 6600aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc acacatgtaa 6660gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt aaaacttatt 6720aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac agccgaagag 6780ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc ttcgcgtcgg 6840cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa tctaccaggg 6900cgcggacaag ccgcgccgtc gccactcgac cgccggcgct gaggtctgcc tcgtgaagaa 6960ggtgttgctg actcatacca ggcctgaatc gccccatcat ccagccagaa agtgagggag 7020ccacggttga tgagagcttt gttgtaggtg gaccagttgg tgattttgaa cttttgcttt 7080gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatct gatccttcaa ctcagcaaaa 7140gttcgattta ttcaacaaag ccgccgtccc gtcaagtcag cgtaatgctc tgccagtgtt 7200acaaccaatt aaccaattct gattagaaaa actcatcgag catcaaatga aactgcaatt 7260tattcatatc aggattatca ataccatatt tttgaaaaag ccgtttc tgt aatgaaggag 7320aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga 7380ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg 7440agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctct gcattaatga 7500atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7560actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 7620gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 7680cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7740ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 7800ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 7860ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 7920agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 7980cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 8040aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga 8100gcgaggtatg taggcggtgc tacagag tgt aatgaaggag 7320aaaactcacc gaggcagttc cataggatgg caagatcctg gtatcggtct gcgattccga 7380ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg ttatcaagtg 7440agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctct gcattaatga 7500atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc 7560actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg 7620gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc 7680cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc 7740ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga 7800ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc 7860ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat 7920agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg 7980cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc 8040aacccggtaa ctggtaacag gattagcaga 8100gcgaggtatg gacacgactt atcgccactg gcagcagcca taggcggtgc tacagag ttc ttgaagtggt ggcctaacta cggctacact 8160agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 8220ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8280cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8340 ttc ttgaagtggt ggcctaacta cggctacact 8160agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt 8220ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag 8280cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg 8340

tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8400aggatcttca cctagatcct tttgatccgg aattaattcc tgtggttggc atgcacatac 8460aaatggacga acggataaac cttttcacgc ccttttaaat atccgattat tctaataaac 8520gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 8580aaggcgggaa acgacaatct gatcatgagc ggagaattaa gggagtcacg ttatgacccc 8640cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc aacgctgcag 8700gaattggccg cagcggccat ttaaatcaat tgggcgcgcc gaattcgagc tcggtac 8757 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa 8400aggatcttca cctagatcct tttgatccgg aattaattcc tgtggttggc atgcacatac 8460aaatggacga acggataaac cttttcacgc ccttttaaat atccgattat tctaataaac 8520gctcttttct cttaggttta cccgccaata tatcctgtca aacactgata gtttaaactg 8580aaggcgggaa acgacaatct gatcatgagc ggagaattaa gggagtcacg ttatgacccc 8640cgccgatgac gcgggacaag ccgttttacg tttggaactg acagaaccgc aacgctgcag 8700gaattggccg cagcggccat ttaaatcaat tgggcgcgcc gaattcgagc tcggtac 8757

Claims (22)

1.一种转化大豆细胞或组织的方法,包括:(a)通过以下步骤从大豆种子制备外植体:(i)从所说的大豆种子中去除下胚轴;(ii)去除一个子叶及其邻近的腋芽,保留附于剩下子叶上的初生叶;以及(iii)从所说的保留子叶上去除一部分初生叶,由此产生初生叶基部;以及(b)将所说的外植体和含有至少一个待掺入到一个或多个大豆细胞的基因组中的目的核酸的农杆菌共培养。 1. A method for transformation of soybean cells or tissue, comprising: (a) from soybean seeds explants prepared by the following steps: (i) removing from said hypocotyls of soybean seeds; (ii) removing a cotyledon and adjacent axillary buds, to retain the remaining primary leaves attached to the cotyledons; and (iii) removing a portion of said primary leaves from the cotyledons retained, thereby generating primary leaf base; and (b) the said explants and Agrobacterium genome a nucleic acid containing at least one to be incorporated into one or more co-cultured soybean cells.
2.权利要求1的方法,还包括在含有选择剂的培养基中培养至少一个形成的芽。 The method of claim 1, further comprising at least one culturing shoots formed in a medium containing the selection agent.
3.权利要求2的方法,其中所说的至少一个目的核酸包括选择标记基因。 The method of claim 2, wherein said at least one nucleic acid of interest comprises a selectable marker gene.
4.权利要求3的方法,其中所说的选择标记基因是磷酸甘露糖异构酶基因。 The method of claim 3, wherein said selection marker gene is mannose phosphate isomerase gene.
5.权利要求4的方法,其中所说的选择剂是甘露糖。 The method of claim 4, wherein said selection agent is mannitol.
6.权利要求4的方法,其中与所说的农杆菌共培养是在甘露糖存在的情况下进行的。 The method of claim 4, wherein said Agrobacterium and co-cultivation is carried out in the presence of mannose.
7.权利要求2的方法,还包括从所说的初生叶基部诱导芽形成。 The method of claim 2, further comprising inducing said primary leaves from the base of the bud formation.
8.权利要求7的方法,其中芽形成是通过在含有芽诱导激素的培养基中培养所说的初生叶基部诱导的。 The method of claim 7, wherein said bud formation by culturing in a medium containing hormones buds induced in the primary leaf base.
9.权利要求8的方法,其中所说的芽诱导激素包括生长素、细胞分裂素和赤霉素中的至少一种。 9. The method of claim 8, wherein said at least one shoot inducing hormones include auxins, cytokinins and gibberellins in.
10.权利要求9的方法,其中所说的生长素选自由IAA、NAA和IBA组成的组。 10. The method set forth in claim 9, wherein said auxin selected from the group consisting of IAA, NAA and IBA thereof.
11.权利要求9的方法,其中所说的细胞分裂素选自由苄氨基嘌呤(BAP)、thidiazuron、激动素和异戊烯基腺嘌呤组成的组。 11. The method set forth in claim 9, wherein said cytokinin is selected from the group consisting of benzylaminopurine (BAP), thidiazuron, kinetin, and isopentenyl adenine thereof.
12.权利要求7的方法,其中芽形成诱导包括去除个或多个附着于子叶上的初生分生组织、次生分生组织和腋生分生组织。 12. The method of claim 7, wherein inducing bud formation comprising removing one or more attached to the primary cotyledon meristem, axillary meristem and secondary meristem.
13.权利要求7的方法,还包括选择转化的芽。 13. The method of claim 7, further comprising selecting transformed shoots.
14.权利要求13的方法,还包括将选择的转化芽再生为大豆植物。 14. The method of claim 13, further comprising selecting transformed shoots regenerated into soybean plants.
15.权利要求1的方法,其中所说的大豆种子是成熟种子。 15. The method of claim 1, wherein said seed is a soybean seed maturation.
16.权利要求1的方法,其中所说的大豆种子是未成熟种子。 16. The method of claim 1, wherein said seed is a soybean immature seeds.
17.权利要求1的方法,其中所说的大豆种子是已发芽种子。 17. The method of claim 1, wherein said seed is a soybean seed has germinated.
18.一种生产稳定转化的大豆植物的方法,包括:(a)通过以下步骤从大豆种子制备外植体:(i)从所说的大豆种子中去除下胚轴;(ii)去除一个子叶及其邻近的腋芽,保留附着在剩下子叶上的初生叶;以及(iii)从所说的保留子叶上去除每一个初生叶的一部分,从而产生一对初生叶基部;(b)将所说的外植体和含有待掺入到大豆细胞基因组中的目的核酸的农杆菌共培养;(c)从每个初生叶基部诱导芽的形成;(d)在含有选择剂的培养基中至少培养一个形成的芽。 18. A method of stably transformed soybean plants produced, comprising: (a) from soybean seeds explants prepared by the following steps: (i) removing from said hypocotyls of soybean seeds; (ii) removing one cotyledon axillary buds and adjacent, attached to the remaining cotyledon retained on the primary leaves; and (iii) removing a portion of each of said primary leaves from the cotyledons retained thereby produce a pair of primary leaf base; (b) the said explants and Agrobacterium tumefaciens containing the nucleic acid to be incorporated into the genome of soybean cells in co-culture; (c) inducing the formation of primary shoots from each leaf base; (d) culturing in a medium containing a selection agent at least a bud formation. (e)选择转化的芽;以及(f)将选择的转化芽再生为大豆植物。 (E) selecting transformed shoots; and (f) selecting the transformed shoots regenerated into soybean plants.
19.从按照权利要求1的方法转化的大豆细胞或组织再生的转基因大豆植物。 19. The method of conversion according to claim 1 from soybean cells or tissue regeneration of transgenic soybean plants.
20.权利要求19的转基因植物生产的转基因种子。 Transgenic seeds of transgenic plants as claimed in claim 19 to 20. A production.
21.从按照权利要求18方法转化的大豆细胞或组织再生的转基因大豆植物。 21. regenerated from soybean cell or tissue according to claim 18 The method of transformation transgenic soybean plants.
22.权利要求21的转基因植物生产的转基因种子。 Transgenic seed of transgenic plants to produce 22. 21 claims.
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